Eimeria maxima 165 rDNA probes

ABSTRACT

Unique species-specific Eimeria maxima DNA probes comprising divergent DNA sequences are disclosed. The probes are complementary to a small subunit ribosomal RNA gene of Eimeria maxima.

RELATED U.S. APPLICATION DATA

This application is a continuation-in-part of application Ser. No.07/706,628, filed May 29, 1991, abandoned, which is incorporated hereinby reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1, A and B. Single strand nucleotide sequence for E. acervulinasmall subunit rRNA gene. (SEQ ID NO:24)

FIG. 2, A and B. Single strand nucleotide sequence for E. brunetti smallsubunit rRNA gene. (SEQ ID NO:25)

FIGS. 3, A and B. Single strand nucleotide sequence for E. maxima smallsubunit rRNA gene. (SEQ ID NO:26)

FIGS. 4, A and B. Single strand nucleotide sequence for E. mitis smallsubunit rRNA gene. (SEQ ID NO:27)

FIGS. 5, A and B. Single strand nucleotide sequence for E. necatrixsmall subunit rRNA gene. (SEQ ID NO:28)

FIGS. 6, A and B. Single strand nucleotide sequence for E. praecox smallsubunit rRNA gene. (SEQ ID NO:29)

FIGS. 7, A and B. Single strand nucleotide sequence for E. tenella smallsubunit rRNA gene. (SEQ ID NO:30)

FIG. 8. Species-specific hybridization to genomic DNA isolated frompurified preparations of Eimeria, showing the specificity of the E.tenella probe.

FIG. 9. Species-specific hybridization to genomic DNA isolated frompurified preparations of Eimeria, showing that the Eimeria probeshybridize to both nonprecocious laboratory isolates and vaccine strains.

FIG. 10. Species-specific detection of Eimeria in the intestinal mucosaof infected chickens.

FIG. 11. Species-specific detection of Eimeria in the intestinal mucosaof heptavalent infected chickens.

FIGS. 12A-R. Multiple nucleotide sequence alignment for chicken Eimeriausing the sequences in FIGS. 1-7. (SEQ ID NO:24-30)

FIG. 13. DNA dot blot analysis using total RNA and species-specificoligonucleotide probes.

FIG. 14. DNA dot blot analysis using total RNA and species-specificoligonucleotide probes.

FIG. 15. Design of species-specific oligonucleotide probes.

FIG. 16. Direct fecal oocysts DNA target in probe hybridization/parasitequantitation assay.

BACKGROUND OF THE INVENTION

Live coccidiosis vaccines (LCV) consisting of particles composed of animmunogenic dose of oocysts from precocious isolates of chicken Eimeriaspecies embedded in a firm gel matrix are well known in the art, asexemplified by U.S. Pat. Nos. 4,544,548, issued Oct. 1, 1985; 4,552,759issued Nov. 12, 1985; 4,752,475 issued on Jun. 21, 1988; 4,863,731issued Sep. 5, 1989 and Patent Cooperation Treaty, InternationalPublication No. WO 85/00752. Evaluation of live coccidiosis vaccinecharacteristics such as viability of each of the species included in thevaccine is paramount to the production and use of the vaccine. Inaddition, any assay used to determine viability must also besemi-quantitative so that the immunogenic efficacy of each species inthe particle can be predicted.

Viability of chicken Eimeria oocysts can only be credibly assessed byexpansion or reproduction in the natural host, as no efficient in vitromodels are available. The ability to detect parasites in the intestinalepithelia and mucosa of vaccinated birds, the target tissue for theseprotozoa, verifies that the organisms are in fact capable of penetratingthe intestinal epithelium and intracellular development. Detection ofoocysts shed in feces indicates that the inoculum contains fullycompetent parasites capable of traversing the entire life cycle.

Historically the Eimeria species (spp.) have been classified accordingto a range of parameters, including morphology, type of pathologyinduced, immunological specificity, characteristic life cycles andbiochemical markers (Joyner and Long, Avian Path. 3, 145-157 [1974]);Shirley, In: McDougold, Joyner and Long, Eds., Research in AvianCoccidiosis, Athens, Ga.: University of Georgia, pp. 13-35 [1985]).However these methods of speciation are tedious and are notquantitative. Furthermore, no single method can unequivocallydifferentiate all species. Infectivity assays for multivalent livecoccidiosis vaccines require unequivocal speciation, semi-quantitationand a stream-lined procedure, owing to the anticipated short half lifeof the vaccine preparation. Existing methodologies do not satisfy theserequirements.

The ribosomal RNA (rRNA) gene loci harbor a wealth of information thathas been successfully used to establish phylogenetic relationships amongand within eukaryotic kingdoms (Hasegawa et al., J. Mol. Evol. 22: 32-80[1985]). Ribosomal RNA genes from protozoa including Toxoplasma gondii(Johnson et al., Exp. Parasitol. 63: 272-278 [1987]), members of thegenus Plasmodium (Dame and McCutchan, J. Biol. Chem. 258: 6984-6990[1983], Langsley et al., Nucleic Acids Res. 11: 8703-8717 [1983]) andEimeria spp. (Ellis and Blumstead, Parasitol. 101: 1-6 [1990]); Johnsonet al., System. Parasitol. 18: 1-8 [1991]) have been cloned andcharacterized towards this end. An extension of this type of analysis inPlasmodium (McCutchan et al., Mol. Biochem. Parasitol. 28: 63-68 [1988])resulted in the design of species-specific oligonucleotide probesderived from the nucleotide sequence of areas within the small subunitrRNA gene.

OBJECTS OF THE INVENTION

It is accordingly, an object of the present invention to preparepurified Eimeria species DNA encoding small subunit ribosomal RNA(ssrRNA) genes free of other Eimeria nucleic acids and other cellularconstituents. A further object is to insert the ssrRNA DNA into suitablevectors, transform an appropriate host with the vector and determine thenucleotide sequence of the DNA. Another object is to provide uniquespecies-specific phylogenetically divergent segments of the ssrRNA geneswhich are used as probes for individual species. Another object is toprepare oligonucleotides complimentary to the divergent regions. Anotherobject is to use the unique probes and assay to quantitate and/oridentify each Eimeria species in a mixture of Eimeria species. A furtherobject is to use the unique probes in a method to quantitate therelative levels of each of multiple Eimeria species in infected hosttissue.

SUMMARY OF THE INVENTION

Unique species-specific Eimeria maxima DNA probes comprising divergentDNA sequences are disclosed. The probes are complementary to a smallsubunit ribosomal RNA gene of Eimeria maxima.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to assays and species-specificidentification probes which allows unequivocal speciation of multipleEimeria species, semi-quantitation of the concentration of each speciesand a shortened time period for determining these parameters.

The following techniques have been used to identify deoxyribonucleicacid (DNA) probes that are specific for each of multiple species ofEimeria that are used to prepare a multivalent coccidiosis vaccine. DNAprobe as used herein refers to a DNA sequence or segment, frequentlylabeled with radioactive isotopes, that is used to identify a gene. ADNA segment as used herein refers to a sequence of nucleotides betweenabout 2 bases and about 2 kb (kilobases) in length. The Eimeria speciesinclude, but are not limited to, E. acervulina, E. brunetti, E. maxima,E. mitis, E. necatrix, E. praecox and E. tenella. An extended list ofEimeria species can be found in Patent Cooperation Treaty, PublicationNo. WO 85/00752. The small subunit rRNA gene from any or all of theEimeria species is cloned and sequenced by the process described herein.Comparative analysis of these nucleotide sequences illustrates multiplesegments within the sequence which are highly conserved across broadphylogenetic lines, as well as regions which are divergent even within agenus (i.e. species-specific). A conserved sequence refers to a DNAsegment in a gene that has not changed in the course of evolution, whilea divergent sequence refers to DNA segment that has changedconsiderably. Divergent sequences vary considerably in the length of theDNA segment which has changed. In the procaryotic genus Francisella,species can be differentiated by a single base difference in theirssrRNA genes, Forsman et al., Appl. Environ. Microbiol. 949-955 (1990).Trypanosomes, on the other hand, contain unique DNA segments in theirssrRNA genes which are several hundred bases long, Dams et al., NucleicAcids Res. 165: r87-r173, (1988). The unique divergent sequences serveas ideal probes for identification of specific species within the genusEimeria. Deoxyribooligonucleotides (single strands of DNA) correspondingto divergent sequences are synthesized, used as hybridization probes andact as effective species-specific reagents.

An assay of this type must be sufficiently sensitive so as to be able todetect the expansion or reproduction of a very small oocyst inoculum. Inother models DNA hybridization probes have been successfully used toquantitate parasite load in infected hosts. For example, exoerythrocyticforms (EEF) of Plasmodium berghei have been assayed in genomic DNAprepared from rat liver extracts using a repetitive plasmodial DNA probe(Ferreira et al., Mol. Biochem. Parasitol. 12: 103-109 [1986]). Morerecently, oligonucleotide probes derived from rRNA sequences have beenemployed to quantitate EEF of Plasmodium yoelii in RNA prepared from thelivers of infected mice (Arreaza et al., Exp. Parasitol. 72: 103-105[1991]). Similarly, any assay for a live coccidiosis vaccine must becapable of detecting Eimeria sequences contained within a total nucleicacid preparation (whether it be RNA or DNA) from chicken intestinalepithelia and mucosa. Because the Eimeria sequences represent such asmall percentage of the genetic information in the extract, directhybridization to DNA is not sufficiently sensitive to detect the vaccineoocyst dose for each species. Because of the biological amplification ofrRNA sequences within cellular RNA pools, hybridization of the Eimeriaspecies-specific oligonucleotide probes to RNA preparations fromintestinal epithelia and mucosa is one way in which this assay andoligonucleotide probes of this invention can be used. Genomic DNAprepared from oocysts shed in the feces of vaccinated birds can also becharacterized as a hybridization target for the oligonucleotide probes.This source of parasite nucleic acid is relatively free of host geneticmaterial and so in effect represents an enriched hybridization target.

Enzymatic amplification of ssrRNA gene sequences in genomic DNA preparedfrom intestinal epithelia and mucosa is a novel alternative approach toenrichment which in the end allows for increased sensitivity in thisunique assay. Using the polymerase chain reaction (PCR; Saiki et al.,Science 239: 487-491 [1988]) and primers which efficiently hybridize toeukaryotic small subunit rRNA genes, it has been possible to selectivelyamplify each of the ssrRNA gene units or fragments thereof within thegenomic DNA prepared from the intestinal epithelia and mucosa ofinfected chickens. Primer as used herein refers to a relatively shortoligonucleotide that specifically attaches to a region ofsingle-stranded template and is necessary to form the starting point forreverse transcriptase to copy adjacent sequences of RNA (mRNA, rRNA andtRNA), or for DNA polymerase to synthesize complementary-strand DNA. Aprimer can also be used with specific polymerases to producecomplementary-strand synthesis with single-stranded genomic DNA, i.e.polymerase chain reaction. Complementary base pairing as used herein isdefined as the linking of bases on double-stranded nucleic acidsaccording to the base-pairing rules which are well known in the art. Acomplementary base sequence is a base sequence in a nucleic acid strandthat is related to the base sequence in another strand by the samebase-pairing rules. This includes Eimeria rRNA genes, chicken rRNA genesas well as rRNA genes derived from any other eukaryotic organism thatmight be represented in the chicken intestine. Amplification isselective in the sense that only small subunit rRNA genes are enrichedas a result of the PCR reaction, but nonspecific because each smallsubunit rRNA gene is enriched to the same extent. The PCR amplificationproducts are quantitated using a fluorescent dye binding assay (Labarcaand Paigen, Anal. Biochem. 102: 344-352 [1980]) and equivalent amountsof amplified DNA fragments are denatured and immobilized on a supportmembrane.

The species-specific oligonucleotide probes are then used inhybridization reactions to determine the presence or absence of each ofthe species of Eimeria in the amplified PCR product and therefore in theintestine of the vaccinated chickens. Hybridization reactions aredefined as a reaction which results in the formation of adouble-stranded molecule by complementary base-pairing between twosingle-stranded DNA molecules, or a single-stranded DNA molecule and anRNA molecule. A control hybridization probe, derived from a portion ofthe sequence contained within the PCR product in a region which isconserved in all eukaryotic small subunit rRNAs, is used to normalizethe amount of denatured and immobilized DNA on the filter. Standardsusing genomic DNA prepared from individual species of Eimeria as PCRtemplates are included on each hybridization filter. These are used toconstruct a standard curve and will also serve as hybridizationspecificity standards. Radioactive emissions from the respective filtersare quantitated using the Molecular Dynamics PhosphorImager (Johnston etal., Electrophoresis 11: 355-360 [1990]).

The following method is used to clone Eimeria small subunit ribosomalRNA (rRNA) genes. It is intended that the following method is not theonly process for cloning the Eimeria small subunit rRNA genes and thatothers known in the art can be used. Oocysts from laboratory strains ofE. acervulina, E. brunetti, E. maxima, E. mitis, E. necatrix, E. praecoxand E. tenella are propagated by oral infection of broiler chickens.Eimeria tenella oocysts are isolated from the cecal contents of chickensat about 5-7 days post-infection. The cecal contents are physicallydisrupted in a Waring Blender, in distilled water and digested withpepsin. Following digestion, debris is removed by centrifugation indistilled water. The remaining Eimeria species are individually isolatedfrom fecal collections about 3-8 days following infection. The feces isdiluted about ten fold in distilled water and then the contents arepassed through a sieving device. A series of passes through screens ofdecreasing size functionally removes a considerable amount of fecaldebris. Partially pure oocyst fractions for each Eimeria species arethen collected by flotation in about 2.2M sucrose (Jackson, Parasitol.54: 87-93 [1964]), and further treated by incubation in sodiumhypochlorite at a concentration of 5.25%, in water at about 40° C. forabout 10 minutes. The sodium hypochlorite is removed by several washesin sterile phosphate buffered saline (PBS) at about pH 7.6 to obtainpurified, sterile oocysts. Depending upon the species, oocysts areallowed to sporulate in a shaking water bath for about 24 to about 60hours at about 20° C. (Edgar, Trans. Am. Micr. Soc. 62: 237-242 [1954])in PBS or sterile water. Following sporulation, oocysts are washedseveral times in PBS.

Sporulated oocysts are disrupted by shaking with 3 mm sterile glassbeads. Beads are added to the oocyst suspension and the mixture is mixedvigorously on a Vortex mixer for about 2 minutes. Periodically theextent of breakage is assessed microscopically. When approximately 50%breakage has occurred, the glass beads are allowed to settle and thesample above the beads is removed and mixed with an equal volume ofPercoll (Pharmacia). The disrupted oocysts are subjected tocentrifugation at about 2,000 to about 5,000×g for about 10 min at about4° C. to pellet the enriched sporocyst fraction. Unbroken oocysts form alayer on top of the 50% Percoll and are removed, washed in PBS, mixedwith glass beads and mixed again as described above. This procedure isperformed repeatedly (3-4 times) until very few unbroken oocysts remainfollowing Percoll fractionation. Sporocyst pellets are combined andwashed several times in PBS.

Sporocysts are then diluted in 0.01M Tris (pH 8.0), 0.2M NaCl to aconcentration of approximately 10⁸ per ml and the suspension is adjustedto about 1% sodium dodecyl sulfate (SDS) and about 10 mM EDTA whichresults in membrane lysis. The released genomic DNA is solubilized bydigestion with Proteinase K (150 μg/ml) for approximately 30 minutes atabout 65° C. Genomic DNA is extracted twice with buffer equilibratedphenol (about pH 7.6), twice with a mixture of phenol/chloroform/isoamylalcohol at about 25:24:1, and twice with chloroform/isoamyl alcohol atabout 24:1. The final aqueous phase is dialyzed overnight in 10 mM Tris(pH 8.0), 10 mM NaCl, 10 mM EDTA (pH 8.0). RNA which has co-purifiedwith the DNA is selectively removed from the dialysate by digestion withheat inactivated RNase A used at a concentration of about 150 μg/ml. Thesamples are incubated for about 1 hour at about 37° C. The RNase andother residual protein is removed by a secondary digestion withProteinase K (about 150 μg/ml, for about 30 minutes at about 37° C.).The genomic DNA is then successively extracted with organic solvents asdescribed above. The final aqueous phase is precipitated with about 0.1volumes of about 3M sodium acetate and about 2.5 volumes of about 100%ethanol. Glycogen is added to 20 μg/ml to act as carrier. The pelletsare washed twice with about 70% ethanol. The genomic DNA pellet is airdried by inversion and is then suspended in about 10 mM Tris.HCl (pH7.6), 1 mM EDTA buffer (TE) or distilled water at a concentration ofabout 5-8×10⁸ sporocyst equivalents/ml and quantitated by absorbence at260 nm. An aliquot of DNA is then analyzed by agarose gelelectrophoresis to confirm (i) the spectrophotometric generatedconcentration, (ii) the lack of residual RNA, and (iii) it's highmolecular weight integrity.

The ribosomal RNA (rRNA) gene loci harbor a wealth of information thathas been successfully used to establish phylogenetic relationships amongand within eukaryotic kingdoms (Hasegawa et al., J. Mol. Evol. 22: 32-80[1985]). Sequences of the small subunit rRNA from a number of highlydivergent organisms have recently been compiled (Dams et al., NucleicAcids Res. 16S: r87-r173 [1988], Neefs et al., Nucleic Acids Res. 18S:2237-2317 [1990]). Comparative analysis of these nucleotide sequencesidentifies areas with dramatic sequence similarities and other areasthat are characterized by considerable sequence drift or divergence.Regions close to both the 5'- and 3'-ends of the consensus small subunitrRNA (ssrRNA) sequence with near identity in the eukaryotic kingdom werechosen. Consensus sequence is defined as a sequence of nucleotidesderived from a large set of observed similar sequences in a specificregion of a nucleic acid. Oligonucleotide primers corresponding to thesesequences were chosen:

    ______________________________________                                        5'-ACCTGGTTGATCCTGCCAG-3' ERIB 1                                              SEQ ID NO: 1                                                                  5'-CTTCCGCAGGTTCACCTACGG-3'                                                                             ERIB 10                                             SEQ ID NO: 2                                                                  ______________________________________                                    

The oligonucleotides were synthesized using an Applied Biosystems 380Binstrument and purified as per the manufacturer's recommendations. TheERIB 1 (SEQ ID NO:1) primer represents a consensus sequence less than 10nucleotides from the 5'-end of eukaryotic ssrRNA genes. The ERIB 10 (SEQID NO:2) primer is the inverse complement to a consensus sequencelocated approximately 20 nucleotides from the 3'-end of eukaryoticssrRNA genes. Taken together, these two oligonucleotides span the vastmajority of the ssrRNA gene sequence. It is intended that the ERIB 1 andERIB 10 primers are not the only primers that can be used to amplify thessrRNA genes or selected fragments thereof. With the knowledge of thepresent invention one could probably devise other primers which couldaccomplish the intended goal.

ERIB 1 (SEQ ID NO:1) and ERIB 10 (SEQ ID NO:2) are used as a primer pairin the polymerase chain reaction (PCR, Saiki et al., Science 239:487-491 [1988]) with the intention of selectively amplifying the ssrRNAgenes contained within the genomic DNA preparation of each Eimeriaspecies as described above. Genomic DNA is quantitated using afluorescent dye binding assay (Lebarca and Paigen, Anal. Biochem. 102:344-352 [1980]) and diluted in distilled water to a final concentrationof about 2.5 ng/μl for use as the PCR template. A 10 X reaction bufferconsisting of about 100 mM Tris-HCl (about pH 8.3), about 500 mM KCl,about 15 mm MgCl₂, about 0.01% gelatin is prepared as well as about 100mM stocks of Tris-HCl (about pH 7.6) buffered dATP, dCTP, dGTP and dTTP.Initially, the reaction mix cocktail is prepared by mixing the followingcomponents at these final concentrations in this specific order: water,dATP, dCTP, dGTP and dTTP (each at about 200 μM), about 1 X reactionbuffer, about 1 μM of each of the two oligonucleotide primers (ERIB 1and ERIB 10) (SEQ ID NO:1, SEQ ID NO:2), and about 1.25 U Taq DNApolymerase. The reaction mixture is assembled in dedicated PCR reactiontubes by combining about 90 μl of the reaction cocktail with about 10 μl(25 ng) of genomic DNA. The reaction is overlayed with approximately 50μl of light mineral oil and then placed into a Perkin Elmer Cetus DNAthermal cycler programmed as follows:

about 35 cycles each composed of (i) about 94° C. for about 60 secondsto denature, (ii) about 50° C. for about 90 seconds to anneal, and (iii)about 72° C. for about 120 seconds for polymerization

about one cycle at about 72° C. for about 10 minutes for extension

A 5 μl aliquot of the amplification reaction product is subjected toagarose gel DNA electrophoresis in TAE buffer (40 mM Tris-acetate, 2 mMethylenediaminetetraacetic acid [EDTA]) along with DNA size standards. Acharacteristic band approximately 1.8 kb in length, whose size isroughly predicted by analogy to other eukaryotic ssrRNA genes, suggeststhat ERIB 1 (SEQ ID NO: 1) and ERIB 10 (SEQ ID NO:2) faithfullyhybridized to the Eimeria ssrRNA genes and that Taq DNA polymerasesynthesized a reaction product by extension from the 3'-ends of theseprimers.

By definition, the ends of the 1.8 kb PCR products correspond to theinput oligonucleotides and should be blunt. However, Taq DNA polymeraseis prone to adding single non-template-directed nucleotides, inparticular dATP, to the 3'-end of duplex PCR products (Clarke, NucleicAcids Res. 16: 9677-9686 [1988]). In order to increase cloningefficiency, the ends of the PCR products are "polished" to blunt-ends bythe action of either T4 DNA polymerase or the Klenow fragment ofbacterial DNA polymerase. Reaction products are extracted once withphenol, once with a phenol/chloroform/isoamyl alcohol mix and once withchloroform/isoamyl alcohol as described earlier. DNA is precipitatedwith sodium acetate/ethanol and the pellet is washed twice with 70%ethanol. For the Klenow fragment reaction, the DNA (about 1-10 μg) issuspended in about 15 μl of water and mixed with about 2 Ill of 1OX nicktranslation buffer (about 0.5M Tris-Cl [pH 7.2], 0.1M MgSO₄, 1 mMdithiothreitol, 500 μg/ml bovine serum albumin [BSA Pentax Fraction V]),and about 2 μl of a 1.25 mM solution of all four dNTPs and about 1 μl(=5 Units) Klenow. The reaction is conducted at about 14° C. for about 1hour and is terminated by heating at about 65° C. for about 10 minutes.The polished 1.8 kb DNA products are passed over a G 25 column,extracted once with phenol, and twice with chloroform/isoamyl alcohol asdescribed earlier. The DNA is precipitated with sodium acetate/ethanoland the pellet is washed twice with about 70% ethanol. The DNA isresuspended in about 36 μl of water and mixed with about 4 μl of 0.2MTris.HCl (pH 9.5), 10 mM spermidine, 1 mM EDTA. This reaction mixture isincubated at about 70° C. for about 5 minutes and subsequently rapidlychilled on ice. To the above 40 μl are added 5 μl of 10X blunt endkinase buffer (0.5M Tris-Cl [pH 9.5], 0.1M MgCl₂, 50 mM dithiothreitol,50% glycerol), and about 5μl of a 10 mM solution of ATP and 2 μl (=20U)of T4 polynucleotide kinase. The reaction is conducted at about 37° C.for about 30 minutes and is terminated by the addition of about 2 μl of0.5M EDTA. The reaction mixture is brought to about 100 μl with TEbuffer and the reaction products are extracted once with phenol, oncewith phenol/chloroform/isoamyl alcohol mix and once withchloroform/isoamyl alcohol as described previously. DNA is precipitatedwith sodium acetate/ethanol and the pellet is washed twice with about70% ethanol, as above. The DNA is resuspended in about 20 μl of waterand quantitated by absorbence at 260 rum.

The polished 1.8 kb DNA products are then agarose gel purified to removeresidual oligonucleotide primers and nonspecific PCR products. Gelslices containing the bands of interest are excised, melted and the DNAeluted using Geneclean II (BIO 101 Inc., Vogelstein and Gillespie, Proc.Natl. Acad. Sci. USA 16: 615-619 [1979]) as per the manufacturer'sinstructions. Eluted DNA products are then quantitated by absorbence at260 run.

A phagemid cloning vector pUC120 (Vieria, Bireplicon Filamentous Phagesand the Production of Single Stranded Plasmid DNA. Ph.D. thesis,University of Minnesota [1989]) is cut at it's unique Sma I site in thepolylinker. Other suitable cloning vectors include but are not limitedto the pGEM-Zf series (Promega Corporation) and the pBluescript IIseries (Stratagene Cloning Systems). Cutting is monitored by analyticalagarose gel electrophoresis. The linearized DNA is then extracted withorganic solvents, precipitated and washed with 70% ethanol as describedearlier. The 5'-end of each strand of the plasmid is phosphatased withcalf intestinal phosphatase (CIP) to decrease the frequency of anautoligation event. This is accomplished by mixing the linearizedplasmid about 10 μg with 5 μl of 10 X CIP buffer (about 0.5M Tris-HCl,pH 9.0, about 10 mM MgCl₂, about 1 mM ZnCl₂, about 10 mM spermidine) andabout 1 μl (1 Unit) of CIP in a final 50 μl reaction volume. Thereaction is conducted for about 15 minutes at about 37° C. and thenabout 15 minutes at about 56° C. A second aliquot of CIP is then addedand the reaction is repeated as above. The reaction is terminated by theaddition of about 40 μl of H₂ O, about 10 μl of about 10 X STE buffer(about 100 mM Tris-HCl, pH 8.0, about 1M NaCl, about 10 mM EDTA), about2.5 μl of about 20% SDS and heated at about 68° C. for about 15 minutes.The linearized, phosphatased vector is then extracted, precipitated andwashed as above.

Ligation of the gel purified ssrRNA gene PCR products into the blunt SmaI site within the pUC120 polylinker is then conducted. Approximately 100ng of linearized vector is mixed with an equimolar amount of therespective PCR products in a 20μl reaction mixture which, in addition iscomposed of about 66 mM Tris-HCl pH 7.6, about 5 mM MgCl₂, about 5 mMdithiothreitol, about 1 mM ATP. The reaction is initiated by theaddition of T4 DNA ligase (about 400 units) and proceeds for about 12-16hours at about 14° C.

Bacterial cells are rendered competent and capable of uptake of foreignDNA by the following method. A predetermined volume (about 2 ml pertransformation reaction) of sterile 2X YT bacterial media (about 16 gbactotryptone, about 10 g yeast extract, about 5 g NaCl per liter) isinoculated with a single colony of Escherichia coli MV1184 and grownwith vigorous mixing at about 37° C. until it reached an optical densityof about 0.6 at about 600 run. Other suitable bacterial hosts includebut are not limited to MN522, JM101, TB1 and XL1-Blue. The bacterialcells are collected by centrifugation at about 1000×g, at about 4° C.,for about 5 minutes. The resulting cell pellet is gently suspended inone-half of the original culture volume with sterile CaCl₂, about 50 mM.The suspension is then placed on ice for about 20 minutes and the cellsare again collected by centrifugation. The cells are then gentlysuspended D in one-tenth volume of sterile 50 mM CaCl₂. The bacterialsuspension is then kept at 4° C. for 16-24 hours.

From the 20 μl ligation reaction mixture about 2 μl and about 18 μlaliquots are dispensed to sterile polypropylene tubes. Approximately 100μl of competent bacteria are added to each of the tubes containing theligation reactions (as well as the appropriate ligation andtransformation controls) and these are placed on ice for 40 minutes.After this, the bacteria are "heat-shocked" by incubation at about 42°C. for 90 seconds and then allowed to recover for approximately 5minutes at room temperature. Each transformation tube is then platedonto a 2X YT agar plate which contains ampicillin at a concentration ofabout 50 mg/l for the selection of bacteria harboring plasmids and forplasmid maintenance. Plates are incubated in an inverted positionovernight at 371 C.

Bacterial clones harboring plasmids are selected by their ability togrow on plates in the presence of ampicillin. Single colonies are usedto inoculate about 5 ml of 2X YT/AMP (i.e., 2X YT media containingampicillin at 50 mg/l) and these cultures are grown overnight at about37° C. with vigorous shaking. Approximately 1.5 ml of the culture ispoured off into an Eppendorf tube and collected by centrifugation in anEppendorf centrifuge for at least 1 min; the remainder of the culture isstored at about 4° C. to serve as a genetic stock. The media above thebacterial pellet is aspirated off and the pellet is suspended by mixingin about 100 μl of a cold, freshly prepared solution of about 50 mMglucose, about 10 mM EDTA, about 25 mM Tris-HCl (pH 8.0), about 4 mg/mllysozyme. This mixture is incubated at room temperature for about 5minutes. Then about 200 μl of a cold, freshly prepared solution,composed of about 0.2N NaOH and about 1% SDS is added to each tube,mixed gently by inversion, and put on ice for about 5 minutes. About 150μl of a cold, freshly prepared solution containing about 6 ml of about5M potassium acetate, about 1.15 ml of glacial acetic acid and about2.85 ml distilled water is added to each tube. The contents are gentlyvortexed and this mixture is stored on ice for about 5 minutes. Thecellular debris is collected by centrifugation in an Eppendorfcentrifuge for 10 minutes at about 4° C. and the supernatant fluid isextracted one time with phenol/chloroform/isoamyl alcohol (about25:24:1). Plasmid DNA and cellular RNA are precipitated from the finalaqueous phase with the addition of two volumes of room temperature 100%ethanol. A pellet is collected by centrifugation for about 5 minutes atroom temperature; the pellet is washed one time with 70% ethanol andthen dried briefly. The nucleic acid pellet is then suspended in about50 μl of TE containing about 20 μg of DNase-free RNase per ml andincubated for about 15-30 minutes at about 37° C. to quantitativelyeliminate cellular RNA. Aliquots of about 10 μl are then cut tocompletion with Hind III and Eco R1 (each at approximately 20 units) ina buffer composed of about 50 mM NaCl, about 100 mM Trim-HCl (pH 7.5)and about 5 mM MgCl₂ at about 37° C. for about 60 min. The restrictionenzyme reaction products are fractionated by agarose gel electrophoresisalong with known DNA size markers to identify those plasmids whichcontained the appropriate inserts. Those recombinant plasmids whichcontain the predicted 1.8 kb insert are then cut with a secondrestriction enzyme (usually Pst I) to verify; (i) that only a singlecopy of the insert is contained within the plasmid, and (ii) to scorefor orientation of the insert DNA with respect to the bacterialpromoter. This is accomplished by removing a second 10 μl aliquot fromthe remaining 40 μl of RNase-digested bacterial nucleic acid and cuttingit in a buffer composed of about 100 mM NaCl, about 10 mM Trim-HCl (pH7.5), about 10 mM MgCl₂ with approximately 20 units of Pst I for about60 D minutes at about 37° C. Again, the restriction enzyme digests areresolved by agarose gel electrophoresis.

Clones containing inserts of the appropriate size were then sequencedusing the dideoxy sequencing protocol (Sanger et al J. Mol. Biol. 143:161-178 [1980]). Single stranded phagemid sequencing templates using K07helper phage were generated exactly as described by Vieria (BirepliconFilamentous Phages and the Production of Single Stranded Plasmid DNA.Ph.D. thesis, University of Minnesota [1989]). Other commerciallyavailable helper phage for the generation of single-stranded templatesfrom phagemid clones include R408 (Promega Corporation for use with thephagemid pGEM-Zf series and bacterial hosts MN522 or JM101) or VCSM13and R408 (Stratagene Cloning Systems for use with the pBluescript IIphagemid series and bacterial hosts XL1-Blue or NM522). Alternatively,double stranded sequencing templates were also used for dideoxysequencing. These were prepared according to the method of Chen andSeeburg (DNA 4:165-170 [1985]). Sequencing reactions were conductedusing a specifically engineered form of T7 DNA Polymerase (Tabor andRichardson, Proc. Natl. Acad. Sci. USA 84: 4767-4771 [1987]). Thisenzyme is available commercially from Pharmacia LKB Biotechnology or asSequenase DNA Polymerase (United States Biochemical Corporation).Reactions were conducted as per the respective manufacturer'sspecifications and the reaction products were resolved by denaturingpolyacrylamide gel electrophoresis (Sanger et al J. Mol. Biol. 143:161-178 [1980]).

Examples of isolated and purified genes encoding the small subunitribosomal RNA of Eimeria species are shown in FIGS. 1-7. The ssrRNA genenucleotide sequences are compared to determine the conserved anddivergent regions of the sequences. Divergent regions are identifiedfollowing comparison and exemplified by the probes illustrated inTable 1. It is intended that the present invention include all of thedivergent DNA regions of the ssrRNA genes of Eimeria species. Thedivergent regions are further defined as DNA sequences of about 1 toabout 50 or about 1 to about 100 nucleotides in length that are notconserved within the organisms that make up the genus Eimeria. It ispreferred that the divergent species-specific sequences are found withinthe ssrRNA of the following Eimeria species: Eimeria species such as: E.acervulina, E. tenella, E. maxima, E. necatrix, E. praecox, E. mitis, E.brunetti. The divergent sequences are identified by comparing thenucleotide sequences shown in FIGS. 1-7.

Table 1 represents the similarity matrix for the seven Eimeria speciesrepresented in FIGS. 1-7. This data was calculated using a computerprogram entitled PILEUP (GCG Software Package, Devereux, Haeberli andSmithies (1984 and A Comprehensive Set of Sequence Analysis Programs forthe VAX. Nucleic Acids Research 12(1); 387-395) using the entiresequence as formatted in FIG. 12. Fundamentally the program does a baseby base comparison of all possible pairs of sequences. The diagonalrepresents a self comparison which is 100% identity. The analysisreveals that the chicken Eimeria are a closely related group. The mostsimilar pair is represented by E. tenella and E. necatrix whose ssrRNAsequences are 99.3% similar. From another view, this pair has 0.7%-dissimilar nucleotide sequences, which over the entire length ofapproximately 1750 bases implies about 12 nucleotide differences. Themost dissimilar pair is represented by E. tenella and E. mitis which are96.4% similar, which implies about 63 nucleotide differences. Thus on aglobal basis the ssrRNA genes in the chicken Eimeria are very similar.Fortunately the differences which do exist seem to be clustered formingdivergent regions, as seen in FIG. 12. If all or a majority of thenucleotide differences were found in one region then oligonucleotideswhich were very dissimilar could be made which would bespecies-specific. Since the positions of the nucleotide differences arenot highly concentrated, the unique oligonucleotides disclosed hereinwould superficially look very similar in some cases having about 2nucleotide differences out of a total of about 20 nucleotides. It isthis similarity in sequence which has necessitated the use of highlystringent hybridization conditions. By highly stringent hybridizationconditions we mean, that conditions (salt concentrations andhybridization and wash temperatures) are such that only perfect hybridsare formed (i.e. all bases in the oligonucleotide hybridization probeperfectly base pair or bond with the PCR amplified fragment). Since wehave consistently used the prehybridization, hybridization and washprotocols described previously we have used the temperature of thehybridization and subsequent washes as the main criteria for stringency.Hybridization and wash temperatures are typically about 3° to about 5°C. below the duplex melting temperature (Tm), where Tm is thetemperature at which 50% of the total number of possible duplexes areannealed under standardized conditions. It is understood that the Tm isdependent upon the salt concentrations used and that changes in thehybridization and wash buffers could dramatically change thehybridization and wash temperatures needed to ensure speciesspecificity.

                  TABLE 1                                                         ______________________________________                                        Similarity Matrix for Avian Eimeria                                           (full length sequences)                                                       Species Ea      Eb      Emx   Emt   En    Ep   Et                             ______________________________________                                        Acervulina                                                                            --                                                                    Brunetti                                                                              97.8    --                                                            Maxima  96.9    97.1    --                                                    Mitis   97.7    97.2    96.3  --                                              Necatrix                                                                              97.4    96.5    95.5  96.5  --                                        Praecox 98.5    97.9    97.5  97.5  97.5  --                                  Tenella 97.5    96.5    96.1  96.4  99.3  97.4 --                             ______________________________________                                    

Table 2 illustrates examples of divergent segment probes useful for thespecific identification of Eimeria. The probes listed in Table 3 derivefrom areas of nucleotide sequence within the small subunit ribosomal RNAgenes which diverge among species and so, using appropriatehybridization and wash conditions (i.e., high stringency), arespecies-specific. Minor changes in the sequence of these probes (e.g.:deletion or addition of nucleotides from the ends), will not necessarilyeliminate the species-specific feature especially if subtle changes inthe hybridization temperature (T_(H)) are similarly incorporated as perthe following equations: T_(h) =T_(m) -5° C.=2° C.(A-T bp)+4° C.(G-Cbp)-5° C. (Suggs et al., In D. D. Brown (ed.), ICN-UCLA Symp. Dev. Biol.Using Purified Genes. Academic Press, Inc. N.Y. Vol. 23, pp. 683-693[1981]) and Tm=ΔH/(ΔS+R×ln(C/4))-273.15° C. (Freier et al., Proc. Natl.Acad. Sci. USA 83: 9373-9377[1986]). It is understood that the inventionwill also include oligonucleotides which are inverse complements of thesequences in Table 1. The inverse sequences represent perfectlysatisfactory species-specific hybridization probes against DNA targets.

The following general PCR amplification oligonucleotide priomers werechosen for E. brunetti:

    ______________________________________                                        5'-AAACTTAAAGGAATTGACGG-3'                                                                              ERIB 7                                              SEQ ID NO: 44                                                                 5'-CGGTGTGTACAAAGGGCAGG-3'                                                                              ERIB 8                                              SEQ ID NO: 45                                                                 ______________________________________                                    

Each of these oligonucleotides is derived from conserved domains ofssrRNA genes and accordingly, are general PCR amplification primers. Theprimers span a length of approximately 508 nucleotides corresponding tonucleotide position 1240 to 1748 in the full length E. brunetti sequence(see FIG. 2B). These two oligonucleotides were used as primers in a PCRreaction which targeted E. brunetti genomic DNA as the amplificationsubstrate using conditions described earlier for the full lengthproducts. The resulting PCR reaction product was cloned into thebacterial plasmid vector pUC120 as described above. Bacterial clonesharboring recombinant plasmids with the appropriate size insert wereidentified and two of these were sequences using the Sanger chaintermination method as described above. The nucleotide sequence of theseclones was identical and is entered as Table 2 entitled E. brunettifragment 4. The nucleotide sequence for the E. brunetti specifichybridization probe pEB4e-rc (SEQ ID NO:36) is complementary tonucleotide positions 224 to 244 in E. brunetti fragment 4, Table 2.

                                      TABLE 2                                     __________________________________________________________________________    Eimeria brunetti fragment 4                                                   __________________________________________________________________________    AAACTTAAAG                                                                              GAATTGACGG                                                                              AGGGGCACCA                                                                              CCAGGCGTGG                                                                              40                                    AGCCTGCGGC                                                                              TTAATTTGAC                                                                              TCAACACGGG                                                                              GAAACTCACC                                                                              80                                    AGGTCCAGAC                                                                              ATGGGAAGGA                                                                              TTGACAGATT                                                                              GATAGCTCTT                                                                              120                                   TCTTGATTCT                                                                              ATGGGTGGTG                                                                              GTGCATGGCC                                                                              GTTCTTAGTT                                                                              160                                   GGTGGAGTGA                                                                              TCTGTCTGGT                                                                              TAATTTCGAT                                                                              AACGAACGAG                                                                              200                                   ACCTTGGCCT                                                                              GCTAAATAGG                                                                              GTCGGTGACT                                                                              TTGGTTACCG                                                                              240                                   TATCGCTTCT                                                                              TAGAGGGACT                                                                              TTGCGTGTCT                                                                              AACGCAAGGA                                                                              280                                   AGTTTGAGGC                                                                              AATAACAGGT                                                                              CTGTGATGCC                                                                              CTTAGATGTT                                                                              320                                   CTGGGCTGCA                                                                              CGCGCGCTAC                                                                              ACTGATGCAT                                                                              GCAACGAGTT                                                                              360                                   TTTACCTTGA                                                                              CCGACGGGGC                                                                              TGGGTAATCT                                                                              TGTGAGGGTG                                                                              400                                   CATCGTGATG                                                                              GGGATAGATT                                                                              ATTGCAATTA                                                                              TTAGTCTTCA                                                                              440                                   ACGAGGAATG                                                                              CCTAGTAGGC                                                                              GCAAGTCAGC                                                                              ACTTGCGCCG                                                                              480                                   ATTACGTCCC                                                                              TGCCCTTTGT                                                                              ACACACCG            508                                   (SEQ ID NO: 50)                                                               __________________________________________________________________________

Live coccidiosis vaccines are produced using oocysts from attenuatedstrains of Eimeria. An example can include, but is not limited to, sevenor more avian Eimeria species such as: E. acervulina, E. tenella, E.maxima, E. necatrix, E. praecox, E. mitis, E. brunetti. An immunogenicdose of oocysts from each species is combined, beaded in wax and coveredwith gypsum. An immunogenic dose refers to a dosage of each specieswhich in combination prevents coccidiosis induced by one or morespecies. One day old female SPF Leghorn chicks are housed in isolatorcages and given non-vaccine containing feed and water ad libitum untiltwo weeks of age. Feed is removed on the day prior to administration ofthe vaccine. Vaccine beads are weighed and aliquots equivalent to 0.25times, 0.5 times, 1 times, 2 times, 3 times, 5 times and 10 times thevaccine dose are mixed with feed (15 g/chick) and presented to thechicks in groups of from between about eight to about fifteen animals.All vaccine should be consumed within four hours. After the vaccine isfully consumed, vaccine-free feed is administered. A group of about 8 to10 untreated birds are fed regular feed and water ad libitum for theduration of the experimental regimen. One to 3 additional groups ofabout 8 to about 15 birds is dosed by gavage with the the same number ofunencapsulated oocysts (1X, 3X and 10X) and fed vaccine free feed adlibitum. These birds will represent a positive control for infection aswell as serving to check the viability of organisms followingencapsulation since the unencapsulated oocysts will be from the sameproduction batch as those in the vaccine. Three to five days followingadministration of the vaccine or unencapsulated oocysts, intestinalepithelial and mucosal scrapings are prepared from the intestinal wallsof the birds. Total nucleic acids extracted from these scrapings serveas the target or template in this protocol. The relative infectivity ofeach species of Eimeria subsequent to the encapsulation process isestimated based upon the ability to detect an amplification of thenumber of input oocysts. This is accomplished using species-specific ³²P-labeled oligonucleotide hybridization probes. Some of the birds ineach treatment group are sacrificed and used for fecal oocyst countsfrom days four to seven post-infection. Quantitation is based on astandard curve using genomic DNA prepared from cloned vaccine strainoocysts.

Preparation of total nucleic acids is carried out with the followingmethod. It is important to take precautions not to introduce nucleasesinto the process, e.g. when possible use baked glassware or use plasticand autoclaved solutions where appropriate. Chickens are sacrificed 3-5days after receiving a vaccine dose. The intestine and the ceca areremoved, cut along their length and rinsed with tap water. The interiorwall of the intestine and ceca are scraped/stripped with a microscopeslide. Scrapings are transferred to a 50 ml centrifuge tube containingabout 5 to 10 ml of 2X Proteinase K digestion buffer (about 400 mMTrim-HCl, pH 7.6, about 100 mM EDTA, about 1.0 % SDS). The suspension ismixed vigorously on a vortex mixer. About 200 μl of about 5 mg/mlProteinase K is added to the suspension and it is allowed to digest atabout 55° C. for about 3 hours. If viscosity is a problem at this point,add about another 5 ml of digestion buffer. Add about 100 μl of 5 mg/mlProteinase K and continue digestion overnight. Following the overnightdigestion, about 100 μl of 5 mg/ml Proteinase K is added and digestionis continued for up to 24 hours. Remove about 600 μl of the digest to a1.5 ml microfuge tube and extract about twice with about a 1:1 mix ofbuffer equilibrated phenol and chloroform. Then extract with about a24:1 mix of chloroform and isoamyl alcohol. The final aqueous phase maybe stored at -20° C. An aliquot of the final aqueous phase is ethanolprecipitated. In most cases about 200 μl of the final aqueous phase isadded to about 20 μl of 3M sodium acetate (pH 4.6) and then combinedwith about 500 μl of ethanol. The samples are mixed by inversion andplaced in a dry ice ethanol bath for about 20 minutes. The genomic DNAis then collected by centrifugation in an Eppendorf microcentrifuge forabout 15 minutes. The precipitate is washed once with about 70% ethanoland dried in a Speed-Vac. The precipitate is suspended in about 200 μlof deionized water. The amount of DNA in this total nucleic acidpreparation is estimated using bisbenzimide which is a fluorochromewhose properties change when bound to DNA as discussed before. Salmontestes DNA standards from 0 to 20 μg/100 μl TE are made from a stocksolution. Prepare the dilutions in 12×75 mm borosilicate tubes usingsterile tips; change tips between dilutions. Similarly, make 1:10dilutions to a final volume of about 100 μl for each experimental samplein duplicate. A bisbenzimide dye stock at a concentration of about 200μg per ml in sterile water and stored at 4° C. in a dark bottle isstable for up to 6 months. Prior to use, dilute the dye stock 1:200 witha buffer whose composition is about 50 mM sodium phosphate, pH 7.6, 2MNaCl. Add about 2 ml of this to each borosilicate tube with an Eppendorfrepeater pipette, mix and measure directly in a fluoro-colorimeter at anexcitation wavelength of 356 nm and an emission wavelength of 458 nm.Determine the amount of DNA in the experimental samples aftercalibrating the machine with standards.

PCR amplification of ssrRNA sequences from genomic DNA prepared fromchicken intestinal mucosal scrapings is carried out in the followingmanner. Due to the exquisite sensitivity of this technique, extremecaution must be exercised to avoid contamination. Dedicated pipettes,pipette tips, vessels and stock solutions for DNA preparation, reactionassembly and sample analysis are recommended. Ideally, this should bedone in areas separate from other DNA handling. About 200 ng ofexperimental genomic DNA based upon the bisbenzimide assay above is usedas the starting target material. It is critical that this material firstbe ethanol precipitated to remove solvents remaining from theextractions which inhibit the Taq DNA polymerase. Genomic DNA preparedfrom known numbers of purified organisms from each of the species ofEimeria are used to "spike" about 200 ng of chicken hepatic genomic DNA.These will serve as amplification standards and hybridizationspecificity standards. Prepare a daily working solution of Tris-HClbuffered (pH 7.6) deoxynucleoside triphosphates dATP, dCTP, dGTP anddTTP each at about 1.25 mM from about 100 mM stocks stored at -20° C.Prepare a 10x reaction buffer composed of about 100 mM Tris- HCl, pH8.3, about 500 mM KCl, about 15 mM MgCl₂, about 0.01% gelatin andautoclave. This is aliquoted and stored at about -20° C. The reactionmixture is assembled in dedicated PCR reaction tubes in a final volumeof about 100 μl. First, a reaction mixture cocktail is prepared bymixing the following components at these final concentrations in thisspecific order: water, dATP, dCTP, dGTP and dTTP (dNTPs each at about200 μM), 1X reaction buffer, and about 1 μM of each of the twoamplification primers (ERIB 1 and ERIB 2) (SEQ ID NO:1 and SEQ ID NO:3)or any other suitable primers which flank a non-consensus region, mix,and add about 1.25 U Taq DNA polymerase per reaction tube and invert tomix. Other primers include, but are not limited to:

    ______________________________________                                        ERIB 2      TCCCTCTCCGGAATCGGAC                                               (SEQ ID NO: 3)                                                                5 ERIB      CCAGGTCCAGACATGG                                                  (SEQ ID NO: 4)                                                                3 ERIB      CTTGCGCCTACTAGGC                                                  (SEQ ID NO: 5)                                                                5AERIB      GTCGCAAGGCTGAAAC                                                  (SEQ ID NO: 31)                                                               3AERIB      CTTGCGCCTACTAGGC                                                  (SEQ ID NO: 32)                                                               5BERIB      GGGGGGAGTATGGTCTGCAAGGC                                           (SEQ ID NO: 33)                                                               3BERIB      GCATGCATCAGTGTAGCTGCGCG                                           (SEQ ID NO: 34)                                                               ______________________________________                                    

Primer ERIB 1 (SEQ ID NO:1) is used with primer ERIB 2 (SEQ ID NO:2) andprimer 5 ERIB (SEQ ID NO:4) is used with primer 3 ERIB (SEQ ID NO:5).Primer 5AERIB (SEQ ID NO:31) is preferably used with primer 3AERIB (SEQID NO:32) and primer 5BERIB (SEQ ID NO:33) is preferably used withprimer 3BERIB (SEQ ID NO:34), however, any primer begining with 5' couldalso be used with any primer begining with 3'. An aliquot of about 80 μlof the cocktail is then distributed to each reaction tube. Based on thebisbenzimide DNA assay described above, about 200 ng of experimentalgenomic DNA is adjusted to a final volume of about 20 μl with distilledwater and added to the reaction mixture. When amplifying using primersERIB1 (SEQ ID NO:1) and ERIB2 (SEQ ID NO:3), the reactions are run inthe BIOS thermal cycler. Generally the BIOS thermal cycler will beprogrammed as follows:

a) about 3 cycles consisting of about 94° C. for about 1 minute todenature, about 50° C. for about 30 seconds to anneal and about 72° C.for about 45 seconds for polymerization;

b) about 27 cycles consisting of about 94° C. for about 20 seconds todenature, about 50° C. for about 30 seconds to anneal and about 72° C.for about 45 seconds for polymerization;

c) about one cycle at about 72° C. for about 10 minutes.

When amplifying using primer pairs 5AERIB/3AERIB (SEQ ID NO:31)/(SEQ IDNO:32) and 5BERIB/3BERIB (SEQ ID NO:33)/(SEQ ID NO:34), the reactionsare run in the Perkin Elmer Cetus DNA thermal cycler. The reactions areset up as described above for the primer pair ERIB1/ERIB2 (SEQ IDNO:1)/(SEQ ID NO:3), except that after addition of the experimentalgenomic DNA, the reaction is overlayed with approximately 50 μl of lightmineral oil and then placed into the Perkin Elmer Cetus DNA thermalcycler programmed as follows:

a) about 3 cycles consisting of about 94° C. for about 1 minute todenature, about 48° C. for about 1 minute to anneal and about 72° C. forabout 1 minute for polymerization;

b) about 32 cycles consisting of about 94° C. for about 1 minute todenature, about 50° C. for about 1 minute 30 seconds to anneal and bout72° C. for about 2 minutes for polymerization;

c) about one cycle at about 72° C. for about 10 minutes. About 5 μl ofthe reaction product is then assayed for DNA content using a small scalebisbenzimide assay analogous to that described above. Exceptions arethat dilutions are done in microcentrifuge tubes in duplicate, the finalassay volume is about 500 μl, the sample is read in a microcell and thestandard curve is linear from about 5 to 200 ng/ml.

Generally, about 100 ng of the PCR product quantitated as describedabove and adjusted to a final volume of about 100 μl with water, isapplied to Nytran sheets (prewetted in water) in a slot-blot or dot-blotmanifold as described in the manufacturer's specifications (Schleicherand Schuell, Inc.). To each sample is added 1 volume of 1M NaOH. Thesamples are then incubated at about room temperature for about 5 minutesto denature the DNA and neutralized by adding about 1 volume of 1MTris-HCl (pH 7.3). A vacuum is then applied to the apparatus to filterthe samples. Each sample is then rinsed with about 500 μl of 4M ammoniumacetate (pH 6.8). Genomic DNA prepared from purified organisms of eachof the avian Eimeria species is PCR amplified as described previouslyand is used to "spike" chicken hepatic genomic DNA also PCR amplified asdescribed previously. The spiked DNA is also applied to the filters toserve as a species-specific quantitation standard. Appropriate buffercontrols and blank controls are routinely included. Air dry the filtersand bake under vacuum at about 80° C. for about 2 hours.

Oligonucleotide hybridization probes are labeled for quantitativedetermination of parasite viability. The preferred method is to endlabel the oligonucleotides with gamma ³² P-ATP. Other methods known inthe art can also be used. Quantitate and standardize oligonucleotides (1mg/ml=25 A₂₆₀). Add about 5-10 pmoles of oligonucleotide to at least atwo fold molar excess of gamma ³² P-ATP (specific activity >5000Ci/mmol), about 5 μl of 10X kinase buffer(about 0.5 Tris-HCl, about pH7.6, about 0.1M MgCl₂, about 50 mM DTT, about 1 mM spermidine, about 1mM EDTA) and a quantity of water sufficient to achieve a 50 μl reactionvolume following the addition of 20 U of polynucleotide kinase. Incubatethe mixture for about 30 minutes at about 37° C. Stop the reaction bythe addition of about 4 μl of 0.5M EDTA, about 46 μl of TE. Extract oncewith a 1:1 mixture of buffer equilibrated phenol and chloroform. Passthe aqueous phase through a Stratagene push column (Stratagene) as perthe manufacturer's specifications to remove the unincorporated isotopefrom the labeled oligonucleotide.

Prehybridization, hydridization and washes are carried out in thefollowing manner. Prehybridization is done in a buffer whose compositionis about 6X SSPE, 1% SDS, 10X Denhardt's, about 20 to 100 μg per mltRNA, and either with or without 50 μg per ml denatured salmon spermDNA. SSPE consists of about 180 mM NaCl, 10 mM NaH₂ PO₄, 1 mM EDTA. Thebuffer is made and kept at 42° C. until ready for use to keep the SDS insolution. The dry sheet(s) of Nytran are prewetted in 6X SSPE, placed ina polyethylene freezer bag which is heat sealed on three sides. Theheterologous DNA is denatured in a boiling water bath for 10 minutes,quick chilled on ice and mixed with the prehybridization solution (20-40ml depending on the number of sheets of Nytran in the bag). Theprehybridization solution is added to the bag, air bubbles removed, andthe bag is sealed on the fourth edge. The bag is then secured to a glassplate with elastic bands and submerged in a water bath at 42° C. for atleast 3 hours or for as long as overnight. Following prehybridizationthe bag is cut open and the buffer is removed completely. Thehybridization buffer is about 6X SSPE plus about 1% SDS. Hybridizationis done at or about the T_(h) of the desired hybrid. For probes lessthan 25 nucleotides in length, hybridization conditions are determinedusing either of the following formulae:

    T.sub.h =T.sub.m -5° C.=2° C.(A-T bp)+4° C.(G-C bp)-5° C.

(Suggs et al., In D. D. Brown (ed.), ICN-UCLA Symp. Dev. Biol. UsingPurified Genes. Academic Press, Inc. N.Y. Vol. 23, pp. 683-693 [1981])

    Tm=ΔH(ΔS+R×ln(C/4))-273.15° C.

(Freier et al., Proc. Natl. Acad. Sci. USA 83: 9373-9377[1986]).

The end labeled oligonucleotide probe is warmed at 68° C. for 5 minprior to mixing with 10-20 ml (depending on the number of filters perbag; approx. 1-5×10⁶ dpm/ml) of hybridization buffer which is prewarmedat the T_(h). This is poured into the bag, air bubbles are removed andthe bag is resealed. The bag is secured to a glass plate and submergedin a water bath at the T_(h) for at least 12 hours to overnight.Following hybridization, cut open the bag and discard the buffer. Cutthe remaining three sides of the bag and with forceps remove thefilter(s) to a pyrex dish containing the first wash solution. The washesare as follows:

a) about 3 times for 5-10 minutes each in 6X SSPE, 1% SDS at 37° C. withshaking;

b) about one time for 3 min in 1X SSPE, 1% SDS at the Th of the hybrid;

c) about 3-4 times for approx. 5 min each in 6X SSPE at room temperaturewith shaking to remove the SDS.

Wash volumes should be at least 100 ml to cover the filters; use morewith multiple filters. All wash solutions should be prewarmed at therespective temperatures prior to use. Air dry the filters, place in acassette which contains two intensifying screens and expose to X-rayfilm at -70° C. Develop the film after 1-3 days.

Quantitation of hybridization signal is carried out using the MolecularDynamics PhosphorImager (Molecular Dynamics). Dried blots are placedbeneath plastic wrap in the PhosphorImager cassette as per themanufacturer's instructions and exposed to the phosphor screen forapproximately 2 hours for the common hybridization probe and 3-12 hoursfor the specific Eimeria probes. The screen is then scanned with a laserwhich releases the energy captured by the phosphor in the screen. Thereleased energy is quantitated by the machine.

Eimeria RNA can also be isolated and used to determine the presence andconcentration of multiple species of Eimeria in a sample. Isolation ofEimeria RNA from chicken intestines must be carried out with care toavoid degradation of the RNA. One successful protocol is essentially thesame as published in Chirgwin et al., Biochemistry 18(1979) 5294-5299.Mucosal scrapings from chickens vaccinated 3-5 days previously are takenand transferred to a 50 ml centrifuge tube as is described earlier.These scrapings are immediately placed into about 24 ml of about 4Mguanidine thiocyanate, pH 7.0, about 0.5% sodium N-lauroylsarcosine,about 25 mM sodium citrate, about 0.1M 2-mercaptoethanol, and about 0.1%Sigma 30% Antifoam A. The samples are quickly homogenized with aPolytron (Brinkmann) at full speed three times for 20 seconds; betweensamples the Polytron is rinsed 2 times with sterile distilled water. Thesamples are then centrifuged at approximately 8,000 RPM for 10 minutesat about 10° C. in a swinging bucket rotor e.g. JS-13 (Beckman). Thesupernatants are decanted and the pellets are precipitated with about0.6 ml of about 1M acetic acid and about 18 ml of 100% ethanol at -20°C., overnight. The samples are centrifuged again at 8,000 RPM, for 10minutes at 10° C. The pellets are resuspended in about 12 ml ofapproximately 7.5M guanidine hydrochloride, pH 7.0, 25 mM sodiumcitrate, and 5 mM dithiothreitol, shaken vigorously, and heated to 68°C. until dissolved. The samples are precipitated with approximately 0.3ml of 1M acetic acid and about 6 ml of 100% ethanol at -20° C.,overnight. Again the samples are centrifuged, resuspended, andprecipitated as before, except with one-half the previous volumes, i.e.6 ml, 0.15 ml, and 3 ml respectively. The samples are pelleted onceagain, triturated with about 10 ml of room-temperature 95 % ethanol,transferred to baked Corex centrifuge tubes, and repelleted at about10,000 RPM for about 30 minutes at about 10° C. The RNA pellets aredried under vacuum in a Speed-Vac (Savant Instruments), dissolved atabout 68° C. in about 2 ml diethyl pyrocarbonate-treated steriledistilled water, repelleted, re-extracted with about 1 ml diethylpyrocarbonate-treated sterile distilled water, and repelleted again. Theextractions are reprecipitated with about 300 μl of 2M potassiumacetate, about pH 5.0, and about 8 ml of 100% ethanol at -20° C.overnight. The final RNA preparations are pelleted and resuspended inabout 1 ml of diethyl pyrocarbonate-treated sterile water. Absorbancereadings at 260 nm and 280 nm (Beckman spectrophotometer) are taken todetermine RNA concentations; about 3 μg of RNA are then subjected toelectrophoresis on about a 1.2% agarose gel to check the RNA quality,size, and relative concentration. RNA samples can be stored at -70° C.The RNA is treated with DNase which is free of RNase (i.e. RQ1 DNase,Promega) as per manufacturers directions, except digestion is carriedout for about 30-40 minutes at about 37° C. The sample is extracted withabout equal volumes of phenol/chloroform and precipitated with about1/10 volume of about 3M sodium acetate and about 2 1/2 volumes ofethanol at about -70° C. overnight. The RNA pellet is recovered bycentrifugation, washed with about 75% ethanol, dried under vacuum andresuspended in diethyl pyrocarbonate-treated sterile water. Twenty tothirty micrograms of RNA are slotted in duplicate onto Nytran filtersafter denaturing the RNA in 1X denaturing solution (4X denaturingsolution contains about 1 ml of formaldehyde, 56 μl of 1M sodiumphosphate, pH 6.5, and 344 μl of sterile distilled water) at 68° C. forabout 20 minutes. The denatured samples are immediately placed on ice tocool and then immobilized onto Nytran filters with a slot/dot-blotmanifold as per manufacturers directions (BioRad Laboratories, Inc.).The nylon filters are baked at about 80° C. for about 30 to 60 minutes.These filters are then prehybridized, hybridized and washed as permanufacturers specifications (Schliecher and Schuell, Inc.) foroligonucleotide probes for Northern (RNA) transfers. The oligonucleotideprobes are ³² P end labelled as previously described.

Genomic DNA from fecal oocysts can also be isolated and used todetermine the presence and concentration of multiple species of Eimeriain a sample. The feces is diluted about ten fold in distilled water andthen the contents are passed through a sieving device. A series ofpasses through screens of decreasing size functionally removes aconsiderable amount of fecal debris. Partially pure oocyst fractions ofthe Eimeria species are then collected by flotation in about 2.2Msucrose (Jackson, Parasitol. 54: 87-93 [1964]), and further treated byincubation in sodium hypochlorite at a concentration of 5.25%, in waterat about 40° C. for about 10 minutes. The sodium hypochlorite is removedby several washes in sterile phosphate buffered saline (PBS) at about pH7.6 to obtain purified, sterile oocysts. Depending upon the species,oocysts are allowed to sporulate in a shaking water bath for about 24 toabout 60 hours at about 20° C. (Edgar,Trans. Am. Micr. Soc. 62: 237-242[1954]) in PBS or sterile water. Following sporulation, oocysts arewashed several times in sterile PBS.

Sporulated oocysts are disrupted by shaking with 3 mm sterile glassbeads. Beads are added to the oocyst suspension and the mixture is mixedvigorously on a Vortex mixer for about 2 minutes. Periodically theextent of breakage is assessed microscopically. When approximately 50%breakage has occurred, the glass beads are allowed to settle and thesample above the beads is removed and mixed with an equal volume ofPercoll (Pharmacia). The disrupted oocysts are subjected tocentrifugation at about 2,000×g for about 10 min at about 4° C. topellet the enriched sporocyst fraction. Unbroken oocysts form a layer ontop of the 50% Percoll and are removed, washed in PBS, mixed with glassbeads and mixed again as described above. This procedure is performedrepeatedly (3-4 times) until very few unbroken oocysts remain followingPercoll fractionation. Sporocyst pellets are combined and washed severaltimes in PBS.

Sporocysts are then diluted in 0.01 M Tris (pH 8.0), 0.2M NaCl to aconcentration of approximately 10⁸ per ml and the suspension is adjustedto about 1% sodium dodecyl sulfate (SDS) and about 10 mM EDTA whichresults in membrane lysis. The released genomic DNA is solubilized bydigestion with Proteinase K (150 μg/ml) for approximately 30 minutes atabout 55° to 65° C. Genomic DNA is extracted twice with bufferequilibrated phenol (about pH 7.6), twice with a mixture ofphenol/chloroform/isoamyl alcohol at about 25:24:1, and twice withchloroform/isoamyl alcohol at about 24:1. The final aqueous phase isdialyzed overnight in 10 mM Tris (pH 8.0), 10 mM NaCl, 10 mM EDTA (pH8.0). RNA which has co-purified with the DNA is selectively removed fromthe dialysate by digestion with heat inactivated RNase A used at aconcentration of about 150 μg/ml. The samples are incubated for about 1hour at about 37° C. The RNase and other residual proteins are removedby a secondary digestion with Proteinase K (about 150 μg/ml, for about30 minutes at about 37° C.). The genomic DNA is then successivelyextracted with organic solvents as described above. The final aqueousphase is precipitated with about 0.1 volumes of about 3M sodium acetateand about 2.5 volumes of about 100% ethanol. Glycogen is added to 20μg/ml to act as carrier. The pellets are washed twice with about 70%ethanol. The genomic DNA pellet is air dried by inversion and is thensuspended in about 10 mM Tris.HCl (pH 7.6), 1 mM EDTA buffer (TE) ordistilled water at a concentration of about 5-8×10⁸ sporocystequivalents/ml and quantitated by absorbance at 260 nm and/or using theafformentioned bisbenzimide assay. An aliquot of DNA is then analyzed byagarose gel electrophoresis to confirm; (i) the spectrophotometricgenerated concentration, (ii) the lack of residual RNA, and (iii) it'shigh molecular weight integrity.

Equivalent amounts of genomic DNA based on the bisbenzimide assay aredenatured and immobilized on eight identical sheets of Nytran paper forhybridization. Generally, about 100 ng of the genomic DNA quantitated asdescribed above is adjusted to about 100 μl with water, to which isadded about 0.1 volume of about 3M NAOH. This is incubated at about 70°C. for about 30-60 minutes to denature the DNA, cooled at roomtemperature, neutralized by adding about 1 volume of about 2M ammoniumacetate (pH 7.0) and applied to Nytran sheets in a slot-blot or dot-blotmanifold as per the manufacture's specifications (Schliecher andSchuell, Inc.). A vacuum is applied to filter the samples. Genomic DNAprepared from known numbers of purified organisms from each of thespecies of Eimeria is also applied to the filters to serve as aspecies-specific quantitation standard. Appropriate buffer controls andblank controls are routinely included. The filters are air dried andbaked under vacuum at about 80° C. for about 2 hours. Theprehybridization, oligonucleotide hybridization, washes and thequantitation of hybridization is carried out as described above.

The following examples illustrate the invention without, however,limiting the same thereto.

EXAMPLE1 Method For Cloning Eimeria Species Small Subunit Ribosomal RNAGenes

Oocysts from laboratory strains of E. acervulina, E. brunetti, E.maxima, E. mitis, E. necatrix, E. praecox and E. tenella were propagatedby oral infection of broiler chickens. Eimeria tenella oocysts wereisolated from the cecal contents of chickens at 5-7 days post-infection.The remaining Eimeria species were individually isolated from fecalcollections 3-8 days following infection. The cecal contents werephysically disrupted in a Waring Blender, in distilled water anddigested with pepsin. Following digestion, debris was removed bycentrifugation in distilled water. The feces was diluted ten fold indistilled water and then the contents were passed through a sievingdevice. A series of passes through screens of decreasing sizefunctionally removed a considerable amount of fecal debris. Partiallypure oocyst fractions for each of the seven Eimeria species were thencollected by flotation in 2.2 M sucrose (Jackson, Parasitol. 54: 87-93[1964]), and further treated by incubation in sodium hypochlorite at aconcentration of 5.25% in water at 40° C. for 10 minutes. The sodiumhypochlorite was removed by several washes in sterile phosphate bufferedsaline (PBS) at pH 7.6 to obtain purified, sterile oocysts. Dependingupon the species, oocysts were allowed to sporulate in a shaking D waterbath for 24 to 60 hours at 20° C. (Edgar,Trans. Am. Micr. Soc. 62:237-242 [1954]) in PBS or sterile water. Following sporulation, theoocysts were washed several times in sterile PBS.

Sporulated oocysts were disrupted using 3 mm sterile glass beads. Beadswere added to the oocyst suspension and the mixture was mixed vigorouslyon a Vortex mixer for approximately 2 minutes. Periodically the extentof breakage was assessed microscopically. When approximately 50% of thesporulated oocysts were disrupted, the glass beads were allowed tosettle and the sample above the beads was removed and mixed with anequal volume of Percoll (Pharmacia). The disrupted oocysts weresubjected to centrifugation at 2,000×g for 10 min at 4° C. to pellet theenriched sporocyst fraction. Unbroken oocysts forming a layer on top ofthe 50% Percoll were removed, washed in PBS, mixed with glass beads andmixed again as described above. This procedure was performed repeatedly(3-4 times) until very few unbroken oocysts remained following Percollfractionation. Sporocyst pellets were combined and washed several timesin PBS.

Sporocysts were then diluted in 0.01M Tris (pH 8.0), 0.2M NaCl to aconcentration of approximately 10⁸ per ml and the suspension wasadjusted to 1% sodium dodecyl sulfate (SDS) and 10 mM EDTA whichresulted in membrane lysis. The released genomic DNA was solubilized bydigestion with Proteinase K (150 μg/ml) for approximately 30 minutes at65° C. Genomic DNA was extracted twice with buffer equilibrated phenol(pH 7.6), twice with a mixture of phenol/chloroform/isoamyl alcohol at25:24:1, and twice with chloroform/isoamyl alcohol at 24:1. The finalaqueous phase was dialyzed overnight in 10 mM Tris (pH 8.0), 10 mM NaCl,10 mM EDTA (pH 8.0). RNA which had co-purified with the DNA wasselectively removed from the dialysate by digestion with heatinactivated RNase A used at a concentration of 150 μg/ml. The sampleswere incubated for 1 hour at 37° C. The RNase and other residualproteins were removed by a secondary digestion with Proteinase K (150μg/ml, for 30 minutes at 55° C.). The genomic DNA was then successivelyextracted with organic solvents as described above. The final aqueousphase was precipitated with 0.1 volume of 3M sodium acetate and 2.5volumes of 100% ethanol. Glycogen was added to 20 μg/ml to act ascarrier. The pellets were washed twice with 70% ethanol. The genomic DNApellet was air dried by inversion and was then suspended in 10 mMTris.HCl (pH 7.6), 1 mM EDTA buffer (TE) or distilled water at aconcentration of 5-8×10⁸ sporocyst equivalents/ml and quantitated byabsorbance at 260 nm. An aliquot of DNA was then analyzed by agarose gelelectrophoresis to confirm (i) the spectrophotometric generatedconcentration, (ii) the lack of residual RNA, and (iii) its highmolecular weight integrity.

The ribosomal RNA (rRNA) gene loci harbor a wealth of information thathas been successfully used to establish phylogenetic relationships amongand within eukaryotic kingdoms (Hasegawa et al., J. Mol. Evol. 22: 32-80[1985]). Sequences of the ssrRNA from a number of highly divergentorganisms have recently been compiled (Dams et al., Nucleic Acids Res.16S: r87-r173 [1988], Neefs et al., Nucleic Acids Res. 18S: 2237-2317[1990]). Comparative analysis of these nucleotide sequences revealedareas with dramatic sequence similarities and other areas that arecharacterized by considerable sequence drift. Regions close to both the5'- and 3'-ends of the consensus small subunit rRNA (ssrRNA) sequencewith near identity in the eukaryotic kingdom were chosen.Oligonucleotides corresponding to these sequences were chosen:

    ______________________________________                                        5'-ACCTGGTTGATCCTGCCAG-3' ERIB 1                                              SEQ ID NO: 1                                                                  5'-CTTCCGCAGGTTCACCTACGG-3'                                                                             ERIB 10                                             SEQ ID NO: 2                                                                  ______________________________________                                    

The oligonucleotides were synthesized using an Applied Biosystems 380Binstrument and purified as per the manufacturer's recommendations. ERIB1 (SEQ ID NO:1) represents a consensus sequence less than 10 nucleotidesfrom the 5'-end of eukaryotic ssrRNA genes. ERIB 10 (SEQ ED NO:2) is theinverse complement to a consensus sequence located approximately 20nucleotides from the 3'-end of eukaryotic ssrRNA genes. Taken together,these two oligonucleotides span the vast majority of the ssrRNA genesequence.

ERIB 1 (SEQ ED NO:1) and ERIB 10 (SEQ ID NO:2) were used as a primerpair in the polymerase chain reaction (PCR, Saiki et al., Science 239:487-491 [1988]) with the intention of selectively amplifying the ssrRNAgenes contained within the genomic DNA preparation of each of the sevenEimeria species as described above. Genomic DNA was quantitated using afluorescent dye binding assay (Lebarca and Paigen, Anal. Biochem. 102:344-352 [1980]) and diluted to a final concentration of 2.5 ng/μl foruse as the PCR template. A 10X reaction buffer consisting of 100 mMTris-HCl (pH 8.3), 500 mM KCl, 15 mM MgCl₂, 0.01% gelatin was preparedas well as 100 mM stocks of Tris-HCl (pH 7.6) buffered dATP, dCTP, dGTPand dTPP. The reaction mixture was prepared by mixing the followingcomponents at these final concentrations in this specific order: water,dATP, dCTP, dGTP and dTTP (each at 200 μM), 1X reaction buffer, 1 μM ofeach of the two oligonucleotide primers (ERIB 1 and ERIB 10) (SEQ IDNO:1 AND SEQ ID NO:2), and 1.25 U Taq DNA polymerase. The reactionmixture was assembled in dedicated PCR reaction tubes by combining 90 μlof the reaction cocktail with 10 μl (25 ng) of genomic DNA. The reactionwas overlayed with approximately 50 μl of light mineral oil and thenplaced into a Perkin Elmer Cetus DNA thermal cycler programmed asfollows:

35 cycles each composed of (i) 94° C. for about 60 seconds to denature,(ii) 50° C. for about 90 seconds to anneal, and (iii) 72° C. for 120seconds for polymerization;

1 cycle at 72° C. for 10 minutes for extension.

A 5 μl aliquot of the reaction product was subjected to agarose gel DNAelectrophoresis in TAE buffer along with DNA size standards. Acharacteristic band approximately 1.8 kb in length, whose size isroughly predicted by analogy to other eukaryotic ssrRNA genes, suggestedthat ERIB 1 (SEQ ID NO:1) and ERIB 10 (SEQ ID NO:2) actually hybridizedto the Eimeria ssrRNA genes and that Taq DNA polymerase synthesized areaction product by extension from the 3'-ends of these primers.

By definition, the ends of the 1.8 kb PCR products correspond to theinput oligonucleotides and should be blunt. However, Taq DNA polymeraseis prone to adding single non-template-directed nucleotides, inparticular dATP, to the 3'-end of duplex PCR products (J. M. Clarke,Nucleic Acids Res. 16: 9677-9686 [1988]). In order to increase cloningefficiency, the ends of the PCR products were "polished" to blunt-endsby the action of the Klenow fragment of bacterial DNA polymerase.Reaction products were extracted once with phenol, once with aphenol/chloroform/isoamyl alcohol mix and once with chloroform/isoamylalcohol as described earlier. DNA was precipitated with sodiumacetate/ethanol and the pellet was washed twice with 70% ethanol. Forthe Klenow fragment reaction, the DNA (1-10 μg) was suspended in 15 μlof water and mixed with 2 μl of 10X nick translation buffer (0.5MTris-Cl [pH 7.2], 0.1M MgSO₄, 1 mM dithiothreitol, 500 μg/ml bovineserum albumin [BSA Pentax Fraction V]), and 2 μl of a 1.25 mM solutionof all four dNTPs and 1 μl (=5 Units) Klenow. The reaction was conductedat 14° C. for 1 hour and was terminated by heating at 65° C. for 10minutes. The polished 1.8 kb DNA products were passed over a G 25column, extracted once with phenol, and twice with chloroform/isoamylalcohol as described earlier. The DNA was precipitates with sodiumacetate/ethanol and the pellet was washed twice with 70% ethanol. TheDNA was resuspended in 36 μl of water and mixed with 4 μl of 0.2MTris.HCl (pH 9.5), 10 mM spermidine, 1 mM EDTA. This reaction mixturewas incubated at 70° C. for 5 minutes and subsequently rapidly chilledon ice. To the above 40 μl are added 5 μl of 10X blunt end kinase buffer(0.5M Tris-Cl [pH 9.5), 0.1M MgCl₂, 50 mM dithiothreitol, 50% glycerol),and 5 μl of a 10 mM solution of ATP and 2 μl (=20U) of T4 polynucleotidekinase. The reaction was conducted at 37° C. for 30 minutes and wasterminated by the addition of 2 μl of 0.5 M EDTA. The reaction mixturewas brought to about 100 μl with TE buffer and the reaction productswere extracted once with phenol, once with phenol/chloroform/isoamylalcohol mix and once with chloroform/isoamyl alcohol as describedpreviously. DNA was precipitated with sodium acetate/ethanol and thepellet was washed twice with 70% ethanol, as above. The DNA isresuspended in 20 μl of water and quantitated by absorbance at 260 nm.

The polished 1.8 kb DNA products were then subjected to agarose gelelectrophoresis to separate the residual oligonucleotide primers andnonspecific PCR products from the polished 1.8 kb products. Gel slicescontaining the bands of interest were excised, melted and the DNA elutedusing Geneclean II (BIO 101 Inc., Vogelstein and Gillespie, Proc. Natl.Acad. Sci. USA 76: 615-619 1979) as per the manufacturer's instructions.Eluted DNA products were then quantitated by absorbance at 260 nm.

A phagemid cloning vector pUC120 (Vieria, Bireplicon Filamentous Phagesand the Production of Single Stranded Plasmid DNA. Ph.D. thesis,University of Minnesota [1989]) is cut at it's unique Sma I site in thepolylinker. Other suitable cloning vectors include but are not limitedto the pGEM-Zf series (Promega Corporation) and the pBluescript IIseries (Stratagene Cloning Systems). Cutting was monitored by analyticalagarose gel electrophoresis. The linearized DNA was then extracted withorganic solvents, precipitated and washed with 70% ethanol as describedearlier. The 5'-end of each strand of the plasmid was phosphatased withcalf intestinal phosphatase (CIP) to decrease the frequency of anautoligation event. This was accomplished by mixing the linearizedplasmid (about 10 μg) with 5 μl of 10X CIP buffer (0.5 M Tris-HCl pH9.0, 10 MM MgCl₂, 1 MM ZnCl₂, 10 mM spermidine) and 1 μl (1 Unit) of CIPin a final 50 μl reaction volume. The reaction was conducted for 15minutes at 37° C. and then 15 minutes at 56° C. A second aliquot of CIPwas then added and the reaction was repeated as above. The reaction wasterminated by the addition of 40 μl of H₂ O, 10 μl of 10X STE buffer(100 mM Tris-HCl, pH 8.0, 1M NaCl, 10 mM EDTA), 2.5 μl of 20% SDS andheated at 68° C. for 15 minutes. The linearized, phosphatased vector wasthen extracted, precipitated and washed as above.

Ligation of the gel purified ssrRNA gene PCR products into the blunt SmaI site within the pUC120 polylinker was then conducted. Approximately100 ng of linearized vector was mixed with an equimolar amount of therespective PCR products in a 20 μl reaction mixture which, in additionis composed of 66 mM Tris-HCl, pH 7.6, 5 mM MgCl₂, 5 MM dithiothreitol,1 mM ATP. The reaction was initiated by the addition of T4 DNA ligaseand incubated for 12-16 hours at 14° C.

Competent bacterial cells capable of uptake of foreign DNA were preparedby the following method. A predetermined volume (about 2 ml pertransformation reaction) of sterile 2X YT bacterial media (16 gbactotryptone, 10 g yeast extract, 5 g NaCl per liter) was inoculatedwith a single colony of Escherichia coli MV1184 and grown with vigorousmixing at 37° C. until it reached an optical density of 0.6 at 600 nm.Other suitable bacterial hosts include but are not limited to MN522,JM101, TB1 and XL1-Blue. Bacteria were collected by centrifugation at1000×g, at 4° C., for 5 minutes. The resulting cell pellet was gentlysuspended in one-half of the original culture volume with sterile 50 mMCaCl₂ and the suspension was then placed on ice for 20 min. The cellswere again collected by centrifugation and then gently suspended inone-tenth volume of sterile 50 mM CaCl₂. The bacterial suspension wasthen kept at 4° C. for 16-24 hours.

From the 20 μl ligation reaction mixture 2 μl and 18 μl aliquots weredispensed into sterile polypropylene tubes. Approximately 100 μl ofcompetent bacteria were added to each of the tubes containing theligation reactions (as well as the appropriate ligation andtransformation controls) and these were placed on ice for 40 minutes.After this, the bacteria were "heat-shocked" by incubation at 42° C. for90 seconds and then allowed to recover for approximately 5 minutes atroom temperature. Each transformation tube was then plated onto a 2X YTagar plate which contains ampicillin at a concentration of 50 mg/l forthe selection of bacteria harboring plasmids and for plasmidmaintenance. Plates were incubated in an inverted position overnight at37° C.

Bacterial clones harboring plasmids were identified by their ability togrow on plates in the presence of ampicillin. Single colonies were usedto inoculate 5 ml of 2X YT/AMP (i.e., 2X YT media containing ampicillinat 50 mg/l) and these cultures were grown overnight at 37° C. withvigorous shaking. Approximately 1.5 ml of the culture was poured offinto an Eppendorf tube and collected by centrifugation in an Eppendorfcentrifuge for at least 1 minute; the remainder of the culture wasstored at 4° C. and served as a genetic stock. The media above thebacterial pellet was aspirated off and the pellet was suspended byvortexing in 100 μl of a cold, freshly prepared solution of 50 mMglucose, 10 mM EDTA, 25 mM Tris-HCl (pH 8.0), 4 mg/ml lysozyme. Thismixture was incubated at room temperature for 5 minutes. Then 200 μl ofa cold, freshly prepared solution composed of 0.2N NaOH, 1% SDS wasadded to each tube, mixed gently by inversion, and put on ice for 5minutes. To this mixture was added 150 μl of a cold, freshly preparedsolution containing 6 ml of 5M potassium acetate, 1.15 ml of glacialacetic acid, 2.85 ml distilled water. The contents were gently vortexedand this mixture was stored on ice for 5 minutes. The cellular debriswas collected by centrifugation in an Eppendorf centrifuge for 10minutes at 4° l C. and the supernatant fluid was extracted one time withphenol/chloroform/isoamyl alcohol (25:24:1). Plasmid DNA and cellularRNA were precipitated from the final aqueous phase with the addition oftwo volumes of 100% ethanol at room temperature . A pellet was collectedby centrifugation for 5 minutes at room temperature, the pellet waswashed one time with 70% ethanol and then dried briefly. The nucleicacid pellet was then suspended in 50 μl of TE containing 20 μl ofDNase-free RNase per ml and incubated for 15-30 minutes at 37° C. toquantitatively eliminate cellular RNA. Aliquots of 10 μl were then cutto completion with Hind III and Eco R1 (each at approximately 20 units)in a buffer composed of 50 mM NaCl, 100 mM Trim-HCl (pH 7.5),5 mM MgCl₂at 37° C. for 60 min. The restriction enzyme reaction products wereseparated by agarose gel electrophoresis to identify those plasmidswhich contained the appropriate inserts. Those recombinant plasmidswhich contained the predicted 1.8 kb insert were then cut with a secondrestriction enzyme (usually Pst I) to verify; (i) that only a singlecopy of the insert was contained within the plasmid, and (ii) to scorefor orientation of the insert DNA with respect to the bacterialpromoter. This was accomplished by removing a second 10 μl aliquot fromthe remaining 40 μl of RNase-digested bacterial nucleic acid andcleaving it in a buffer composed of 100 mM NaCl, 10 mM Trim-HCl (pH7.5), 10 mm MgCl₂ with approximately 20 units of Pst I for 60 min at 37°C. Again, the restriction enzyme digests were resolved by agarose gelelectrophoresis.

The isolated and purified genes encoding the E. acervulina, E. brunette,E. maxima, E. mitis, E. necatrix, E. praecox and E. tenella smallsubunit ribosomal RNA are shown in FIGS. 1-7 respectively. The sevengene sequences were compared and regions of nucleotide divergence wereidentified. Oligonucleotides complimentary to these divergent regionswere synthesized as described above and were used as hybridizationprobes as described below. Table 4 illustrates the primary divergentsequences for the various species of Eimeria. The sequences listed inTable 3 (except for those in the `common` group) are examples of themost convenient species-specific hybridization probes, i.e. probes whichwere constructed to regions of the ssrRNA genes containing the maximaldiversity of nucleotide sequence, resulting in maximal specificity.

                                      TABLE 3                                     __________________________________________________________________________     Eimeria species                                                                      Probe Name                                                                           Sequence                                                       __________________________________________________________________________    acervulina                                                                            WEaRC  CAGCCCACGCAATTAAGCGCAGGAG                                                                            (SEQ ID NO: 6)                                  PEa4-RC                                                                              GAAGTGATACGATAACCGAAGTT                                                                              (SEQ ID NO: 7)                                  PEa4e-RC                                                                             TACGATAACCGAAGTTACCG   (SEQ ID NO: 35)                         brunetti                                                                              AEb1RC CCCCTTCATAGAAAGGAAGCC  (SEQ ID NO: 8)                                  AEb1aRC                                                                              CCCCTTCAAAGAAGGAAGCC   (SEQ ID NO: 9)                                  PEb4-RC                                                                              TGCGTGACCGAGGTCA       (SEQ ID NO: 10)                                 PEb4e-RC                                                                             GATACGGTAACCAAAGTCACC  (SEQ ID NO: 36)                         maxima  WEmx1RC                                                                              CAAGACTCCACAAGAATTGTG  (SEQ ID NO: 11)                                 PEmx4-RC                                                                             GATACGGTAACCGAGGTCAC   (SEQ ID NO: 12)                                 PEmx4a-RC                                                                            GATACGGTAACCGAGGTCA    (SEQ ID NO: 37)                         mitis   PEmt1RC                                                                              CCAGAGGAGGGCCTATGCG    (SEQ ID NO: 13)                                 PEmt1aRC                                                                             CCAGAGGAGGAGGCCTATGCG  (SEQ ID NO: 14)                                 PEmt4-RC                                                                             TGACCTGGTGACCCAGG      (SEQ ID NO: 15)                         necatrix                                                                              WEn-1RC                                                                              CGTTAAGTGGGTTGGTTTTG   (SEQ ID NO: 16)                                 WEn-1M CAAAACCAACCCACTTAACG   (SEQ ID NO: 38)                                 PEn4-RC                                                                              AAGTGATACAGTAATCGTGAAGTT                                                                             (SEQ ID NO: 17)                         praecox WEp1RC CACCATGACTCCACAAAAGTG  (SEQ ID NO: 18)                                 PEp4-RC                                                                              AGAAGTGATACAGTAACCGAAGTT                                                                             (SEQ ID NO: 19)                                 Pep4d-RC                                                                             TGATACAGTAACCGAAGTTACTG                                                                              (SEQ ID NO: 39)                         tenella WEt1RC CCAAGACTCCACTACAAAGTG  (SEQ ID NO: 20)                                 PEt4-RC                                                                              GTGATACAGTAACCGCAAAGTT (SEQ ID NO: 21)                                 PEt4a-RC                                                                             TACAGTAACCGCAAAGTTACTG (SEQ ID NO: 40)                         common  CommonRC                                                                             AGCCATTCGCAGTTTCACCG   (SEQ ID NO: 22)                                 Common4RC                                                                            AAGGTCTCGTTCGTTATCGA   (SEQ ID NO: 23)                                 Com4A-RC                                                                             GGTCTCGTTCGTTAATCGAA   (SEQ ID NO: 41)                                 COM4B-RC                                                                             CATCACAGACCTGTTATTGCC  (SEQ ID NO: 42)                                 COM4C-RC                                                                             CATAGAACGGCCATGCA      (SEQ ID NO: 43)                         __________________________________________________________________________

Other regions of the ssrRNA genes which can serve the same purpose arelisted in Table 4. An indicator of ssrRNA sequence diversity wasobtained by computer analysis of the sequences illustrated in FIGS. 1-7.The program PRETTY within the GCG (Univ. of Wisconsin) program packagewas used as an example of a multiple sequence alignment program. Theobjective of the algorithm of this program is to maximize the areas ofhomology between the sequences compared by making base by basecomparisons and inserting gaps which correspond to additions ordeletions as necessary to optimize the number of matches. FIG. 12 is anexample of the output generated by `PRETTY` using the sequencesillustrated in FIGS. 1-7. Note that there is an additional line ofsequence termed `consensus.` This is a position by position report onthe homology of the sequences compared. If all seven nucleotides match,a capital letter is used to identify that event. If a single differenceis observed it is denoted by a() in the consensus sequence. It shouldalso be noted that in this `aligned` format, all seven species end upwith a sequence length of 1766 bases, do to the insertion of varioussized gaps. Thus the nucleotide numbering system in FIG. 12 is relativeto the alignment program and program parameters used. Nucleotidesegments of interest in the `aligned` format must be cross referenced tothe absolute sequence numbering system for each individual species.

                  TABLE 4                                                         ______________________________________                                        Regions Of The ssrRNA Gene From Chicken Eimeria Species                       Useful As Species-Specific Hybridization Probe Targets.                       Nucleotide Positions Relative To The `Alignment` In FIG. 12.                  Region      Nucleotide Span                                                   ______________________________________                                        1           106-114                                                           2           154-180                                                           3           189-227                                                           4           257-272                                                           5           636-733                                                           6           932-939                                                           7           1037-1052                                                         8           1062-1068                                                         9           1160-1173                                                         10          1341-1392                                                         11          1487-1529                                                         12          1679-1716                                                         ______________________________________                                    

Areas of the ssrRNA gene from the seven chicken Eimeria species, whichhave diverged during the course of evolution can be identified bycomparing the `consensus` sequence and in particular locating areaswhere dashes cluster (see FIG. 12). Using this type of analysisapproximately 12 regions within the ssrRNA gene from the chicken Eimeriahave been identified which contain sufficient species-to-speciesnucleotide sequence diversity to be useful hybridization probe targets,i.e. regions which will serve as templates for oligonucleotidehybridization probes. Table 5 lists these regions using the `aligned`nucleotide numbering system. Table 5 lists the same regions using theabsolute sequence numbering system for each species as illustrated inFIGS. 1-7. The following table contains the nucleotide position for eachregion of Table 4, for each of the Eimeria species.

                  TABLE 5                                                         ______________________________________                                        Species: Region, (Nucleotide Span)                                            ______________________________________                                        E. acervulina: 1, (106-113); 2, (153-179); 3, (188-215); 4, (254-             267); 5, (631-728); 6, (927-934); 7, (1031-1047); 8, (1057-                   1063); 9, (1155-1168) 10, (1336-1378); 11, (1473-1515); 12,                   (1665-1700).                                                                  E. brunetti: 1, (106-113); 2, (153-179); 3, (188-222); 4, (252-               264); 5, (629-726); 6, (925-932); 7, (1030-1045); 8, (1054-                   1061); 9, (1153-1167); 10, (1334-1375); 11, (1470-1512); 12,                  (1661-1669).                                                                  E. maxima; 1, (106-113); 2, (153-179); 3, (188-226); 4, (256-                 269); 5, (633-730); 6, (929-936); 7, (1034-1049); 8, (1059-                   1065); 9, (1157-1170); 10, (1338-1380); 11, (1475-1517); 12,                  (1667-1702).                                                                  E. mitis: 1, (106-113); 2, (153-179); 3, (188-223); 4, (253-266);             5, (630-725); 6, (923-928); 7, (1026-1041); 8, (1051-1057); 9,                (1149-1161); 10, (1329-1380); 11, (1474-1517); 12, (1667-                     1701).                                                                        E. necatrix; 1, (106-114); 2, (154-180); 3, (188-226); 4, (255-               271); 5, (635-732); 6, (931-938); 7, (1036-1051); 8, (1060-                   1067); 9, (1159-1172); 10, (1340-1384); 11, (1479-1521); 12,                  (1671-1708).                                                                  E. preacox: 1, (106-113); 2, (153-179); 3, (188-223); 4, (253-                266); 5, (630-727); 6, (927-933); 7, (1031-1046); 8, (1056-                   1062); 9, (1154-1168); 10, (1335-1375); 11, (1471-1514); 12,                  (1664-1699).                                                                  E. tenella: 1, (106-114); 2, (154-180); 3, (189-226); 4, (255-                271); 5, (635-732); 6, (931-938); 7, (1036-1047); 8, (1061-                   1067); 9, (1159-1172); 10, 1340-1384); 11, (1479-1521); 12,                   (1671-1708).                                                                  ______________________________________                                    

EXAMPLE 2 Infectivity Assay

Live coccidiosis vaccine lots were produced using oocysts fromattenuated strains of Eimeria. A vaccine was prepared with the followingEimeria species: E. acervulina, E. tenella, E. maxima, E. necatrix, E.praecox, E. mitis, E. brunetti. An immunogenic dose of oocysts from eachspecies was combined, beaded in wax and covered with gypsum. One day oldfemale SPF Leghorn chicks were housed in isolator cages and givennon-vaccine containing feed and water ad libitum until two weeks of age.Feed was removed on the day prior to administration of the vaccine.Vaccine beads were weighed and aliquots equivalent to 0.25X, 0.5X, 1X,2X, 3X, 5X and 10X vaccine dose were mixed with feed (15 g/chick) andpresented to the chicks in groups of 8 to 10 animals. All vaccine wasconsumed within four hours. After the vaccine was fully consumed,vaccine-free feed was administered for the duration of the test. A groupof 8 to 10 untreated birds were fed regular feed and water ad libitumfor the duration of the experimental regimen. One additional group of 8to 10 birds was dosed by gavage with the the same number ofunencapsulated oocysts (1X) and fed ad libitum. These birds representeda positive control for infection and served to check the viability oforganisms following encapsulation since the unencapsulated oocysts werefrom the same production batch as those in the vaccine. Three to fivedays following administration of the vaccine or unencapsulated oocysts,mucosal and epithelial scrapings were prepared from the intestinal wallsof the birds. Total nucleic acids extracted from these scrapings servedas the hybridization target or PCR amplification template in thisprotocol. The relative infectivity of each species of Eimeria subsequentto the encapsulation process was estimated based upon the ability todetect an amplification of the number of input oocysts. This wasaccomplished using the species-specific ³² P-labeled oligonucleotidehybridization probes described in Example 1. Some of the birds in eachtreatment group were saved to monitor fecal oocyst counts from days fourto seven post-infection. Quantitation was based on a standard curveusing genomic DNA prepared from cloned vaccine strain oocysts.

Preparation Of Total Nucleic Acids

Chickens were sacrificed 3-5 days after receiving a vaccine dose. Theintestine and the ceca were removed, cut along their length and rinsedwith tap water. The interior wall of the intestine and ceca were scrapedusing a microscope slide. The scrapings were transferred to a 50 mlcentrifuge tube and processed immediately. Five to 10 ml of 2XProteinase K digestion buffer (400 mM Tris-HCl, pH 7.6, 100 mM EDTA, 1.0% SDS) were added to the scrapings and the suspension was mixedvigorously on a vortex mixer. To the suspension was added 200 μl of 5mg/ml proteinase K and the suspension was allowed to digest at 55° C.for 3 hours. If viscosity was a problem at this point another 5 ml ofdigestion buffer and another 100 μl of 5 mg/ml proteinase K were addedand digestion was continued overnight. Following the overnightdigestion, 100 μl of 5 mg/ml proteinase K was added and digestion wascontinued for up to 3 to 24 hours. Six hundred microliters of the digestwas removed and placed into a 1.5 ml microfuge tube and extracted twicewith a 1:1 mixture of digestion buffer equilibrated phenol andchloroform. The samples were then extracted with a 24:1 mix ofchloroform and isoamyl alcohol. The final aqueous phase was stored at-20° C. An aliquot of the final aqueous phase was ethanol precipitated.In most cases 200 μl of the final aqueous phase was added to 20 μl of 3Msodium acetate (pH 4.6) and then combined with 500 μl of ethanol. Thesamples were mixed by inversion and placed in a dry ice ethanol bath for20 minutes. The genomic DNA was then collected by centrifugation in anEppendorf microcentrifuge for 15 minutes. The precipitate was washedonce with 70% ethanol and dried in a Speed-Vac (Savant). The precipitatewas suspended in 200 μl of deionized water. The amount of DNA in thetotal nucleic acid preparation was estimated using bisbenzimide which isa fluorochrome whose properties change when bound to DNA as mentionedpreviously. Salmon testes DNA standards ranging from 0 to 20 μl/100 μlin TE were made from a stock solution. Dilutions were prepared in 12×75mm borosilicate tubes using sterile tips which were changed betweendilutions. Similarly, 1:10 dilutions were prepared to a final volume of100 μl for each experimental sample in duplicate. A bisbenzimide dyestock at a concentration of 200 μl per ml sterile water was prepared andstored at 4° C. in a dark bottle. Prior to use, the dye stock wasdiluted 1:200 with a buffer whose composition was 50mM sodium phosphate,pH 7.6, 2M NaCl. Two milliliters of this were added to each borosilicatetube with an Eppendorf repeater pipette, mixed and measured directly ina fluoro-colorimeter at an excitation wavelength of 356 nm and anemission wavelength of 458 nm. The amount of DNA in the experimentalsamples was determined after calibrating the machine with theappropriate standards as described by the manufacturer.

PCR Amplification Of Protozoan ssrRNA Sequences From Genomic DNAPrepared From Chicken Intestinal Epithelial and Mucosal Scrapings

Due to the exquisite sensitivity of this technique, extreme caution wasexercised to avoid contamination. Dedicated pipettes, pipette tips,vessels and stock solutions for DNA preparation, reaction assembly andsample analysis were used. Two hundred ng of experimental genomic DNAbased upon the bisbenzimide assay above were used as the starting targetmaterial. This material was first ethanol precipitated to removesolvents remaining from the extractions which inhibit the Taq DNApolymerase. Genomic DNAs prepared from known numbers of purifiedorganisms from each of the species of Eimeria were used to "spike" 200ng of chicken hepatic genomic DNA. These served as amplificationstandards and hybridization specificity standards. A daily workingsolution of Tris-HCl buffered (pH 7.6) deoxynucleoside triphosphatesdATP, dCTP, dGTP and dTTP each at 1.25 mM was prepared from 100 mMstocks stored frozen at -20° C. A 10X reaction buffer composed of 100 mMTris- HCl, pH8.3, 500 mM KCl, 15 mM MgCl₂, 0.01% gelatin was preparedand autoclaved. This was then aliquoted and stored at -20° C. Thereaction mix was assembled in dedicated PCR reaction tubes in a finalvolume of 100 μl. A reaction mix cocktail was prepared by mixing thefollowing components at these final concentrations in this specificorder: water, dATP, dCTP, dGTP and dTTP (dNTPs each at 200 μM), 1Xreaction buffer, and 1 μl of each of the two amplification primers (ERIB1 and ERIB 2) (SEQ ID NO:1 and SEQ ID NO:3), then mixed, and 1.25 U TaqDNA polymerase per reaction tube was added and mixed by inversion. Analiquot of 80 μl of the cocktail was then distributed to each reactiontube. Based on the bisbenzimide DNA assay described above, 200 ng ofexperimental genomic DNA was adjusted to a final volume of 20 μl withdistilled water and added to the reaction mixture. The BIOS thermalcycler was programmed as follows:

a) 3 cycles consisting of 94° C. for 1 minute to denature, 50° C. for 30seconds to anneal and 72° C. for 45 seconds for polymerization;

b) 27 cycles consisting of 94° C. for 20 seconds to denature, 50° C. for30 seconds to anneal and 72° C. for 45 seconds for polymerization;

c) one cycle at 72° C. for 10 minutes.

When using primer pairs 5AERIB/3AERIB (SEQ ID NO:31/SEQ ID NO:32) or5BERIB/3BERIB (SEQ ID NO:33/SEQ ID NO:34), a reaction mixture wasprepared by mixing the following components at these finalconcentrations in this specific order: water, dATP, dCTP, dGTP and dTTP(dNTPs each at 200 μM), 1X reaction buffer, and 1 μM of each of the twoamplification primers (5AERIB [SEQ ID NO:31] and 3AERIB [SEQ ID NO:32]or 5BERIB [SEQ ID NO:33] and 3BERIB [SEQ ID NO:34), then mixed and 1.25U Taq DNA polymerase per reaction tube was added and mixed by inversion.An aliquot of 80 μl of the mixture was then distributed to each reactiontube. Based on the bisbenzimide DNA assay described above, 200 ng ofexperimental genomic DNA was adjusted to a final volume of 20 μl withdistilled water and added to the reaction mixture. The reaction wasoverlayed with approximately 50 μl of light mineral oil and then placedinto a Perkin Elmer Cetus DNA thermal cycler programmed as follows:

a) 3 cycles consisting of 94° C. for 1 minute to denature, 480 C. for 1minute to anneal and 72° C. for 1 minute for polymerization;

b) 32 cycles consisting of 94° C. for 1 minute to denature, 50° C. for 1minute 30 seconds to anneal and 72° C. for 2 minutes for polymerization;

c) one cycle at 72° C. for 10 minutes.

Five μl of the reaction product were then assayed for DNA content usinga small scale bisbenzimide assay analogous to that described above. Thesmall scale assay used dilutions in microcentrifuge tubes in duplicate,with a final assay volume of 500 μl. The samples were read in amicrocell and the standard curve was linear from 5 to 200 ng/ml.

Immobilization Of Nucleic Acids On A Nylon Support In A Slot-blot OrDot-blot Manifold

Generally, 100 ng of PCR product as quantitated above was adjusted to100 μl with water and applied to Nytran sheets (prewetted in water) in aslot-blot or dot-blot manifold as per the manufacturer's specifications(Schleicher and Schuell, Inc.). To each sample was added 1 volume of 1MNaOH. The samples were then incubated at room temperature for 5 minutesto denature the DNA and subsequently neutralized by adding 1 volume of1M Trim-HCl (pH 7.3). A vacuum was then applied to the apparatus tofilter the samples. Each sample was then rinsed with 500 μl of 4Mammonium acetate (pH 6.8). Genomic DNA prepared from purified organismsrepresenting each of the species of chicked Eimeria and subjected to PCRas described previously was used to "spike" chicken hepatic genomic DNAalso subjected to PCR as described previously. The spiked DNA wasapplied to the filters and served as a species-specific quantitationstandard. Appropriate buffer controls and blank slot controls wereroutinely included. The filters were air dried and baked under vacuum at80° C. for 2 hours (optional).

Oligonucleotide hybridization probes (from Example 1) were end labeledwith gamma ³² P-ATP. The oligonucleotides were quantitated andstandardized using the the following formula (1 mg/ml=25 A₂₆₀). Five toten picomoles of oligonucleotide were added to a 50 μl reaction volumecontaining water, 5 μl of 10X kinase buffer (0.5 Tris-HCl, pH 7.6, 0.1MMgCl₂, 50 mM DTT, 1 mM spermidine, 1 mM EDTA), 20U of polynucleotidekinase, and at least two fold molar excess of gamma ³² P-ATP (specificactivity >5000 Ci/mmol). The mixture was incubated for 30 minutes at 37°C. and then stopped by the addition of 4 μl of 0.5M EDTA, 46 μl of TE.The reaction mixture was extracted once with a 1:1 mixture of bufferequilibrated phenol and chloroform and the aqueous phase was passedthrough a Stratagene push column (Stratagene) as per the manufacturer'sspecifications to remove the unincorporated isotope from the labeledoligonucleotide.

Prehybridization, Hydridization And Washes

Prehybridization was carried out in a buffer whose composition was 6XSSPE, 1% SDS, 10X Denhardt's, 100 μl per nil TRNA. The buffer was madeand kept at 42° C. until ready for use to keep the SDS in solution. Thedry sheet(s) of Nytran were prewetted in 6X SSPE, placed in apolyethylene freezer bag which was heat sealed on three sides. Theprehybridization solution (20-40 ml depending on the number of sheets ofNytran in the bag) was added and the bag was sealed on the fourth edgeafter removing the bulk of the air bubbles, secured to a glass platewith elastic bands and submerged in a water bath at 42° C. for at least3 hours or for as long as overnight. Following prehybridization the bagwas cut open and the buffer was removed completely. The hybridizationbuffer was 6X SSPE plus 1% SDS. Hybridization was done at the T_(h) ofthe desired hybrid. For probes less than 25 nucleotides in length,hybridization conditions were determined using the following formula:

    T.sub.h =T.sub.m =5° C.=2° C.(A-T bp)+4° C.(G-C bp)-5° C.

The end labeled oligonucleotide probe was warmed at 68° C. for 5 minprior to mixing with 10-20 ml (depending on the number of filters perbag; approx. 1-5×10⁶ dpm/ml) of hybridization buffer which was prewarmedat the T_(h). This was poured into the bag, air bubbles were removed andthe bag was resealed. The bag was secured to a glass plate and submergedin a water bath at the T_(h) for at least 12 hours to overnight forhybridization. Following hybridization, the bag was cut open and thebuffer was discarded. The remaining three sides of the bag were cut andthe filters were removed with forceps to a pyrex dish containing thefirst wash solution. The washes were as follows:

a) 3 times for 5-10 minutes each in 6X SSPE, 1% SDS at 37° C. withshaking;

b) 1 time for 3 min in 1X SSPE, 1% SDS at the T_(h) of the hybrid;

c) 3-4 times for approx. 5 min each in 6X SSPE at room temperature withshaking to remove the SDS.

Wash volumes were at least 100 ml; more with multiple filters. All washsolutions were prewarmed at the respective temperatures prior to use.The filters were air dried, placed in a cassette, which contained twointensifying screens, and exposed to X-ray film at -70° C. The film wasdeveloped after 1-3 days. Quantitation of hybridization signal wascarried out using the Molecular Dynamics PhosphorImager (MolecularDynamics). Dried blots were placed beneath plastic wrap in thePhosphorImager cassette as per the manufacturer's instructions andexposed to the phosphor for approximately 2 hours for the commonhybridization probe and 3-12 hours for the specific Eimeria probes. Thescreen was then scanned with a laser which releases the energy capturedby the phosphor in the screen. The released energy was quantitated bythe machine.

EXAMPLE 3 Use Of Specific Eimeria Species Small Subunit Ribosomal RNAProbes And Assay

Purified oocysts from multiple strains of each of the seven species ofchicken Eimeria were prepared as described in Example 1. Sporocysts werepurified after disruption of the oocyst shell. Genomic DNA was preparedfrom each population of sporocysts and quantitated using thebisbenzimide assay. Four micrograms of each preparation of genomic DNAwere denatured and immobilized on a Nytran membrane in eight equivalent0.5 ug aliquots. Gloves were worn and forceps used whenever handlingNytran. Generally about 0.5 μg of genomic DNA was adjusted to about 100μl (4 μg/800 μl ) and added to 0.1 volume of 3M NaOH. This was incubatedat about 70° C. for about 30 to 60 minutes to denature the DNA, cooledto room temperature, neutralized by adding about one volume of 2Mammonium acetate (pH 7.0)) and applied to Nytran sheets in a slot-blotor dot-blot manifold as described by the manufacturer (Schleicher andSchuell, Inc). Vacuum was applied to the apparatus to filter thesamples. Appropriate buffer controls and blank slot controls wereroutinely included. The filters were air dried and baked under vacuum atabout 80° C. for about 2 hours. Chicken genomic DNA (ClonetechLaboratories, Inc.) was similarly denatured and immobilized. The eightfilters were prehybridized in individual bags and then hybridized withthe respective species-specific probes (X7) and a probe common to alleukaryotic ssrRNA gene sequences. The common probe used was `common RC`with the following sequence: AGCCATTCGCAGTTTCACCG (SEQ ID NO:22). Thecommon probe was derived from highly conserved sequence segments. Thisis only an example of one of many such probes which could be made forconserved sequences within the ssrRNA gene. It is understood that onlythose sequences which are spanned by the particular PCR primer pair areuseful as probes for that target. These probes could be used tonormalize the signal across broad phylogenetic groups (i.e. Eimeria andGallus). FIG. 8 shows the results generated using the E.tenella specificprobe (WEt1RC) (SEQ ID NO:20). Only those slots in the grid containingE. tenella genomic DNA gave positive hybridization signals or responsewith WEt1RC. The DNA in these slots was derived from a field isolate, alaboratory strain, a precocious isolate (the vaccine strain) and aclonal derivative of the vaccine strain. Each of the four gave roughlyequivalent hybridization signals. This indicates that the hybridizationprobe is species-specific, but not specific for the vaccine strain.

Similar types of experiments designed to verify the species-specifichybridization characteristics for the remaining six Eimeria probes wereconducted and the results from three of these are depicted in FIG. 9.Probes derived from the ssrRNA genes of E.praecox (WEp1RC) (SEQ ID NO:18), E.maxima (WEmx1RC) (SEQ ID NO:11) and E.necatrix (WEn-1M) (SEQ EDNO;38) (from Example 2) are indeed species-specific. As is the case witheach of the Eimeria probes, hybridization to both nonprecociouslaboratory isolates and vaccine strains is roughly equivalent.

Hybridization of the eighth replicate filter with the probe derived fromssrRNA gene nucleotide sequence common to eukaryotes (common RC, SEQ IDNO:22) indicated that an equivalent amount of "hybridizable" genomic DNAwas immobilized in each of the labeled grids.

Groups of two chickens were each dosed by gavage with 2,500 purifiedoocysts of a single species of chicken Eimeria. An additional pair ofbirds did not receive any oocysts. Five days later the birds weresacrificed, the intestinal epithelia and mucosa was scraped and genomicDNA was prepared from this tissue. The resulting DNA was quantitated and200 ng were aliquoted for use as a reaction substrate in the polymerasechain reaction (PCR) along with the PCR amplification primers ERIB 1(SEQ ID NO:1) and ERIB 2 (SEQ ID NO:3). Ten percent of the reactionproduct was then denatured and immobilized on eight identical slot blotgrids. FIG. 10 shows the results from hybridization of one of thesepanels with the E.brunetti specific probe (AEb1RC) (SEQ ID NO:8). Onlythose birds dosed with E.brunetti oocysts gave a positive hybridizationsignal with this probe. These and similar results with the otherhybridization probes not only reconfirmed the species-specific nature ofthe respective hybridization probes but, also and more importantly,indicated a high sensitivity for the detection of an infection stemmingfrom 2500 oocysts.

The vaccine dose however was considerably fewer than 2500 oocysts andwas composed of all seven species of chicken Eimeria. In the nextexperiment an equal number of oocysts from all seven species were mixedtogether and birds were dosed by gavage with a titration of thisheptavalent mixture. The range of the dose titration was from 100oocysts of each of the seven species to 2500 oocysts of each of theseven species. Five days following infection intestinal epithelia andmucosa was scraped and genomic DNA was extracted and quantitated (asdescribed in Example 2). Two hundred nanograms of each sample were usedas reaction substrates in the PCR using the ERIB 1 (SEQ ID NO:1) andERIB 2 (SEQ ID NO:3) amplification primers. The reactions were done intriplicate and the products from these individual reactions wereimmobilized in successive rows in the slot blot manifold as is indicatedin the right hand margin of FIG. 11. In addition, 10 μl (10%), 1 μl (1%)and 0.1 μl (0.1%) of each reaction product was loaded in columns A, Band C respectively. Seven identical filters were prepared and each washybridized with one of the species-specific probes. Results using theE.brunetti specific probe (AEb1RC) (SEQ ID NO:8) are shown in FIG. 11.Importantly, an unequivocal hybridization signal was detected in bird426 which received a dose of 100 oocysts of each species. This resultindicates that the PCR/hybridization assay is sensitive enough to detectan infection in the intestine of a chicken that had received a 1Xvaccine dose (100 oocysts for E.brunetti). Similar results were obtainedwith probes specific for the remaining six species.

FIG. 11 also serves to illustrate that triplicate polymerase chainreactions do not result in equivalent amounts of reaction products,despite starting with an equivalent amount of the same reactionsubstrate. This observation has led us to incorporate twostandardization steps into the assay protocol. First, the productsresulting from the PCR are quantitated in a small scale bisbenzimideassay which consumes only 5% of the reaction. Using this result, 800 ngof product were denatured and immobilized onto Nytran paper in eightequivalent aliquots of 100 ng each. The eighth replicate filter wasroutinely hybridized with the common probe (common RC) (SEQ ID NO:22) toconfirm that an equivalent amount of denatured and immobilizedhybridization target was present in each experimental slot on thefilter.

EXAMPLE 4 Assay Method For Detecting Eimeria Ribosomal RNA WithSpecies-Specific Oligonucleotides

Isolation of Eimeria RNA from chicken intestines was carried out withcare to avoid degradation of the RNA. The protocol is essentially thesame as published in Chirgwin et al., Biochemistry 18: 5294-5299 (1979).Chickens were orally infected with oocysts fom laboratory strains of E.acervulina. E. brunetti. E. maxima. E.mitis. E. necatrix. E. praecox andE. tenella. Five days later the chickens were sacrificed. Theirintestines and ceca were taken out, cut along their length, and rinsedthroughly with running tap water. The interior walls of the intestinesand ceca were scraped with a sterile microscope slide. The mucosalscrapings from each chicken were taken and transferred to a 50 mlcentrifuge tube. These scrapings were immediately placed into 24 ml ofabout 4M guanidine thiocyanate, pH 7.0, 0.5% sodium N-lauroylsarcosine,25 mM sodium citrate, 0.1M 2-mercaptoethanol, and 0.1% Sigma 30%Antifoam A. The samples were quickly homogenized with a Polytron(Brinkmann) at full speed three times for 20 seconds; between samplesthe Polytron was rinsed 2 times with sterile distilled water. Thesamples were then centrifuged at approximately 8000 RPM for 10 minutesat about 10C. in a swinging bucket rotor (JS-13, Beckman). Thesupernatant fluids were decanted and precipitated with 0.6 ml of 1Macetic acid and 18 ml of 100% ethanol at -20° C., overnight. The nextday the samples were centrifuged again at 8000 RPM for 10 minutes at10C. The pellets were resuspended in 12 ml of 7.5M guanidinehydrochloride, pH 7.0, 25 mM sodium citrate, and 5 mM dithiothreitol,shaken vigorously, and heated to 68° C. until dissolved. The sampleswere precipitated with 0.3 ml of 1 M acetic acid and 6 ml of 100%ethanol at -20° C., overnight. Again the samples were centrifuged,resuspended, and precipitated overnight at -20° C. as before, exceptwith one-half the previous volumes, i.e. 6 ml, 0.15 ml, and 3 mlrespectively. The samples were pelleted once again, triturated withabout 10 ml of room-temperature 95% ethanol, transferred to baked Corexcentrifuge tubes, and repelleted at 10,000 RPM for 30 minutes at about10° C. The RNA pellets were dried under vacuum in a Speed-Vac (SavantInstruments), dissolved at 68° C. in 2 ml diethyl pyrocarbonate-treatedsterile distilled water, repelleted, re-extracted with about 1 mldiethyl pyrocarbonate-treated sterile distilled water, and repelletedagain. The extractions were reprecipitated with 300 μI of 2M potassiumacetate, pH 5.0, and 8 ml of 100% ethanol at -20° C. overnight. Thefinal RNA was pelleted and resuspended in 1 ml of diethylpyrocarbonate-treated sterile water. Absorbance readings at 260 nm and280 nm (Beckman spectrophotometer) were taken to determine RNAconcentations; about 3 μg of RNA was then subjected to electrophoresison a 1.2% agarose gel to check the RNA quality, size, and relativeconcentration. RNA samples were stored at -70° C. One milligram of RNAwas subjected to DNase-1 digestion using RQ1 DNase (Promega) for 40minutes at 37° C. as per the manufacturer's specifications and thenprecipitated with 1/10th volume of 3M NaOAc and 2.5 volumes of 100%ethanol. Duplicate samples containing twenty micrograms of RNA weredenatured in 100 μl of 1 X denaturation solution (four timesdenaturation solution contained 1 ml of formaldehyde, 56 μl of 1M sodiumphosphate, pH 6.5, and 344 μl of sterile distilled water.) at 68° C. for20 minutes. The denatured samples were then placed on ice to cool. Thedenatured RNA samples were dotted in duplicate using a Bio-Rad dot-blotapparatus, Nytran filters (S & S), and 10X SSPE. The filters were driedfor one hour in an 80° C. oven. The filters were probed with ³²P-labelled oligos as in example 2. The filters were prehybridized andhybridized as per the manufacturer's specifications (Schleicher &Schull) for RNA hybridizations using oligonucleotide probes and using aT_(h) as specified for each oligo as previously described. The resultsare shown in FIGS. 13 and 14.

FIG. 13 is a composite of five Nytran filters (Schleicher & Schull) onwhich approximately 30 μg of DNase 1 digested total cellular RNA wasspotted. The RNA was derived from duplicate chickens given a heptavalentmixture of oocysts. The filters were processed as described above. Therow labeled `C` was an uninfected chicken control. The rows labeled `lX`and `lOX` represented the vaccine dosage used while the adjacent rowsrepresent duplicate samples. The panel labeled `ERIB2` was a controlpanel to establish equal loading. It was probed with the Erib2oligonucleotide (SEQ ID NO 3) the sequence of which is derived from ahighly conserved region of the ssrRNA genes and which hybridized to boththe infected and uninfected controls. The panel labeled `Eb` was probedwith the oligonucleotide AEblRC (SEQ ID NO: 8) and at the lOX dose afaint E. brunetti signal was seen. The panel labeled `Emx` was probedwith WEmxlRC (SEQ ED NO: 11). A faint E. maxima signal was seen at lOXand clearly seen at the lOX dose. The panel labeled "Ep" was probed withWEplRC (SEQ ID NO: 18) and E. praecox was demonstrated in both the lOXand lOX doses. The panel labeled `Et` was probed with WEtlRC (SEQ ID NO:20) and a faint E. tenella signal was seen at the lX dose, while the lOXdose is clearly seen.

FIG. 14 is similar to FIG. 13 except that only the lOX dose was used anddifferent oligonucleotides were used as hybridization probes. The panellabeled `ERIB2` was probed with the oligonucleotide Erib2 (SEQ ID NO 3)and it hybridized to both infected and uninfected controls with equalintensity. The panel labeled `Eb` was probed with the oligonucleotidePEb4e-RC (SEQ ID NO: 36) and an E. brunetti signal was clearly observed.The panel labeled `Emt` was probed with PEmt4-RC (SEQ ID NO: 15) and E.mitis was detectable at this level. The panel labeled `Emx` was probedwith PEmx4a-RC (SEQ ID NO: 37) and an E. maxima signal was seen. Thepanel labeled `En` was probed with the oligonucleotide PEn4-RC (SEQ IDNO: 17) and an E. necatrix signal was observed. The panel labeled `Ep`was probed with PEp4d-RC (SEQ ID NO: 39) and a faint E. praecox signalwas detected. The panel labeled `Et` was probed with PEt4a-RC (SEQ IDNO: 40) and an E. tenella signal was observed.

EXAMPLE 5 A METHOD FOR DESIGNING SPECIES SPECIFIC OLIGONUCLEOTIDEHYBRIDIZATION PROBES

Once the ssrRNA sequences from all seven avian Eimeria were determinedand aligned nonconserved regions were identified. The sequences withinthe nonconserved regions were analyzed to determine if there weresufficient differences to allow the construction of species specificoligonucleotide hybridization probes. There were three constraints whichwe placed on the design of our hybridization probes. One was that theprobes be species specific. Due to the nature of the assay crosshybridization could not be tolerated. The second was to have a set ofoligonucleotide hybridization probes which had melting temperatures(T_(m)) which were as close as possible to a single temperature so as toallow the use of a single hybridization temperature. This constraint wasmore a matter of convenience than necessity. The last constraint was tomake the oligonucleotides the reverse compliment of the sense strand, sothe the probes could be used for probing either DNA or RNA. Startingwith the sequence for the nonconserved region an oligonucleotide wasfound which had a T_(m) of approximately 60° C. The probe wassynthesized and tested for specificity.

The target DNA for these specificity studies was obtained in thefollowing manner. Genomic DNA from each of the seven Eimeria species wasused as DNA template in the PCR using two amplification primers 5ERIB(SEQ. ID NO: 4) and 3ERIB (SEQ. ID NO: 5). The use of this particularprimer pair is important since they would not produce an amplificationproduct when the DNA temple was derived from either chicken or E. coli,i.e. the primer pair is specific for Eimeria ssrRNA genes. This primerpair flanks the nonconserved region to which the oligonucleotidehybridization probes were designed. The reactions were run in thePerkin-Elmer Cetus DNA thermal cycler. The reactions contained about25ng of Eimeria genomic DNA and were set up as previously described forthis machine. The Perkin-Elmer Cetus DNA thermal cycler was programmedas follows:

a) about 35 cycles consisting of 94° C. for about 1 minute to denature,about 50° C. for about 1.5 minutes to anneal and about 72° C. for about2 minutes for polymerization.

b) about one cycle at about 72° C. for about 10 minutes.

About 5 ml of the reaction mixture is then assayed for DNA content usingthe small scale bisbenzimide assay. About 5 ml of the reaction mixtureis electrophoresed on an about 2% agarose gel to ensure that thereaction produced a single amplification product. About 10 ng of the PCRproduct was adjusted to a final volume of about 100 μl with water, andapplied to Nytran sheets (prewetted in water) in a slot-blot manifold asdescribed in the manufacturer's specifications (Schleicher and Schuell,Inc.). To each sample was added about 1 volume of 1M NaOH. The sampleswere then incubated at about room temperature for about 5 minutes todenature the DNA and were neutralized by adding about 1 volume of 1MTrim-HCl (pH 7.3). A vacuum was then applied to the apparatus to filterthe samples. Each sample was then rinsed with about 500 ml of 4Mammonium acetate (pH 6.8). Appropriate buffer controls and blankcontrols were included. The Nytran sheets were air dried then bakedunder vacuum at about 80° C. for about 2 or more hours. The test probewas labelled for hybridization. The preferred method was to end labelthe test oligonucleotides with gamma ³² P-ATP by methods which have beenpreviously described. Prehybridizations, hybridizations and washes werealso carried out as previously described. Since specificity was themajor issue being addressed, a probe was considered species-specificwhen only the slot containing the targeted species DNA yielded signal.If any other slot gave a signal the probe was not species-specific andnot useful in this context.

FIG. 15 illustrates the development of a species-specific hybridizationprobe for E. acervulina in region 10. The first oligonucleotidesynthesized was pEa4-RC (SEQ. ID NO: 7) which hybridized with the E.acervulina target as well as E. bruneti, E. mitis, E. maxima, and E.praecox. Further analysis revealed that a single base had been omitted(a T at position #12) so this probe was resynthesized to yield pEa4a-RC(SEQ. ID. NO: 46), which hybridized to the target E. acervulina as wellas E. maxima. A dramatic improvement in the species-specificity wasobtained by the addition of one base, however, some cross-hybridizationdid occur. The next oligonucleotide tested was pEa4b-RC (SEQ. ID. NO:47) which not only hybridized to the target E. acervulina but also E.maxima, and E. praecox as well. Oligonucleotide pEa4c-RC (SEQ. ID. NO:48) was synthesized and tested and found to be as good as pEa4a-RC.(SEQ. ID. NO: 46) When pEa4 d-RC (SEQ. ID. NO: 49) was synthesized andtested it also was found to be as good as pEa4a-RC (SEQ. ID. NO: 46).Species-specificity was finally demonstrated when oligonucleotidepEa4e-RC (SEQ. ID. NO: 35) was synthesized and tested. A similarapproach was used to develop species specific hybridization probes forthe other six chicken Eimeria species.

EXAMPLE 6 Direct Hybridization to Genomic DNA Prepared from FecalOocysts as a Method for Parasite Detection and Quantitation

Fecal oocysts from birds infected with a monovalent or a mixed inoculumof Eimeria oocysts were collected. Oocysts were purified from fecalmaterial. Sporocysts were isolated and purified from sporulated oocysts.Methods for collection, purification and sporulation of oocysts, andsubsequent purification of sporocysts have been described in Example 1.The number of sporocysts in each monovalent collection sample werecounted either by Coulter counter or haemocytometer. Genomic DNA wasprepared from a known number of each monovalent population of sporocystsas well as from the mixed population of sporocysts from the heptavalentinfected groups of birds. The isolation of genomic DNA from sporocystsis described in Example 1. Methods for denaturation and immobilizationof genomic DNA on nylon membranes for hybridization are described inExample 3.

Panels I and II in FIG. 16 illustrate typical results which demonstratethe feasibility of this particular method. The two panels areidentically loaded and the order of loading is indicated by the textbetween the panels. Genomic DNA prepared from fecal oocysts ofmonovalent infected birds is immobilized in rows one through thirteen. Atitration of sporocyst equivalents of this genomic DNA from each speciesis loaded in columns A, B and C. However, the absolute number ofsporocyst equivalents differs among the species. For example, there are1.24×10⁶ E. maxima sporocyst equivalents in slots 7A, but only 1.0×10⁶E. tenella sporocyst equivalents in slots 11A. Slots 15A, B and Ccontain a titration of chicken genomic DNA and are included to serve asa negative hybridization control. Slots 17A, 17B, 17C and 18A contain10% of the genomic DNA prepared from an unknown number of sporocystspurified from four separate heptavalent experimental infections.

Filters I and II were prehybridized in individual bags and thenhybridized with the E. maxima (WEmx1RC, SEQ ID NO: 11) and the E.tenella (WEt1RC, SEQ ID NO:20) species specific probes, respectively. Inpanel I hybridization specificity is demonstrated by the observationthat only row 7, which contains the E. maxima DNA target, shows asignificant signal. Moreover, the signal intensity decreases from slot7A to 7B to 7C which correlates with the titration of immobilized targetDNA in these slots. Only one of the four experimental slots (number 19A)containing genomic DNA from heptavalent infected birds hybridized withthe E. maxima probe. The intensity of the signal corresponds to theintensity seen with the signal in slot 7C, or roughly 0.3×10⁶ sporocystequivalents. Since 10% of the entire experimental sample was loaded inslot 19A, we estimate that the total number of E. maxima sporocysts inthe mixed sporocyst population was on the order of 3×10⁶ . The lack ofhybridization to DNA immobilized in slots 17A, 17B and 17C suggests thatthese experimental samples contain less than 1×10⁶ E. maxima sporocystequivalents.

Hybridization specificity with the E. tenella probe is demonstrated inpanel II by the fact that only one of the seven monovalent infectedexperimental samples (row 11) generates a positive signal. Thehybridization signal titrates in a manner which correlates with therelative amount of E. tenella sporocyst genomic DNA equivalentsimmobilized in slots 11A, 11B and 11C. The approximate number ofsporocyst equivalents is indicated by the numbers over these slots. Twoof the four experimental slots (numbers 17C and 19A) that containgenomic DNA from heptavalent infected birds hybridized with the E.tenella probe. By comparison to the hybridization signals in row 11, weestimate that slots, 17C and 19A contain <0.25×10⁶ and 0.5×10⁶ sporocystequivalents, respectively. Since these slots contain 10% of the totalgenomic DNA prepared from the experimental samples, the total number ofE. tenella sporocysts in the mixed sporocyst population was on the orderof <2.5×10⁶ and 5×10⁶, respectively. By analogy, heptavalent infectedexperimental samples corresponding to slots 17A and 17B appear tocontain less than 1×10⁶ E. tenella sporocyst equivalents.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 50                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       ACCTGGTTGATCCTGCCAG1 9                                                        (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       CTTCCGCAGGTTCACCTACGG21                                                       (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       TCCCTCTCCGGAATCGGAC19                                                         (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       CCAGGT CCAGACATGG16                                                           (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       CTTGCGCCTACTAGGC16                                                            (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 bases                                                           (B) TYPE: nucleic acid                                                       (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       CAGCCCACGCAATTAAGCGCAGGAG25                                                   (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi ) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                      GAAGTGATACGATAACCGAAGTT23                                                     (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       CCCCTTCATAGAAAGGAAGCC21                                                       (2) INFORMATION FOR SEQ ID NO:9:                                              ( i) SEQUENCE CHARACTERISTICS:                                                (A) LENGTH: 20 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       CCCCTTCAAAGAAGGAAGCC20                                                        (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      TGCGTGACCGAGGTCA16                                                            (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      CAAGACTCCACAAGAATTGTG21                                                       (2 ) INFORMATION FOR SEQ ID NO:12:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      GATACGGTAACCGAGGTCAC20                                                        (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 bases                                                          (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      CCAGAGGAGGGCCTATGCG19                                                         (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      CCAGAGGAGGAGGC CTATGCG21                                                      (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      TGACCTGGTGACCCAGG17                                                           (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 bases                                                           (B) TYPE: nucleic acid                                                       (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      CGTTAAGTGGGTTGGTTTTG20                                                        (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                       AAGTGATACAGTAATCGTGAAGTT24                                                   (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      CACCATGACTCCACAAAAGTG21                                                       (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 24 bases                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      AGAAGTGATACAGTAACCGAAGTT24                                                    (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      CCAAGACTCCACTACAAAGTG21                                                       (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      GTGATACAGTAACCGCAAAGTT22                                                      (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      AGCCATTCGCAGTTTCACCG20                                                        (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 bases                                                          (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      AAGGTCTCGTTCGTTATCGA20                                                        (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1748 bases                                                        (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      TAGTCATAT GCTTGTCTCAAAGATTAAGCCATGCATGTCT40                                   AAGTATAAGCTTTTATACGGTGAAACTGCGAATGGCTCAT80                                    TAAAACAGTTATAGTTTATTTGATGGTCTCTTTTACATGG120                                   ATAACCATGGTAATTCTATGGCTAATACATGCGCAAGGGC160                                   CTC CTCCTCTGGAGGGGCTGTGTTTATTAGATACAAAACC200                                  AACCCACCTTGTGTGGAGTCTTGGTGATTCATAGTAACCG240                                   AACGGATCGCAGTTGGCTTTCGGGCCCGCGATGGATCATT280                                   CAAGTTTCTGACCTATCAGCTTTCGACGGTAGGGTATTGG3 20                                  CCTACCGTGGCAGTGACGGGTAACGGGGAATTAGGGTTCG360                                   ATTCCGGAGAGGGAGCCTGAGAAACGGCTACCACATCTAA400                                   GGAAGGCAGCAGGCGCGCAAATTACCCAATGAAAACAGTT440                                   TCGAGGTAGTGACGAGAAATAACAATACAGGGCATC TTAT480                                  GCTTTGTAATTGGAATGATGGGAATGTAAAACCCTTCCAG520                                   AGTAACAATTGGAGGGCAAGTCTGGTGCCAGCAGCCGCGG560                                   TAATTCCAGCTCCAATAGTGTATATTAGAGTTGTTGCAGT600                                   TAAAAAGCTCGTAGTTGGATTTCTGTCGT GGTCAGCCTGC640                                  GCTGCCCGTATGGGTGTGCGCGTGGTTTGCCCTCGGCTTT680                                   TTTCTGGTAGCCTCCTGCGCTTAATTGCGTGGGCTGGTGT720                                   TCCGGAACTTTTACTTTGAGAAAAATAGAGTGTTTCAAGC760                                   AGGCTTGTCGCCCTGAATACT GCAGCATGGAATAATAAGA800                                  TAGGACCTCGGTTCTATTTTGTTGGTTTCTAGGACCAAGG840                                   TAATGATTAATAGGGACAGTTGGGGGCATTCGTATTTAAC880                                   TGTCAGAGGTGAAATTCTTAGATTTGTTAAAGACGAACTA920                                   CTGCGAAAGCATTT GCCAAGGATGTTTTCATTAATCAAGA960                                  ACGACAGTAGGGGGTTTGAAGACGATTAGATACCGTCGTA1000                                  ATCTCTACCATAAACTATGCCGACTAGAGATAGGGAAATG1040                                  CCTACCTTGGCTTCTCCTGCACCTCATGAGAAATCAAAGT1080                                  CTCT GGGTTCTGGGGGGAGTATGGTCGCAAGGCTGAAACT1120                                 TAAAGGAATTGACGGAGGGGCACCACCAGGCGTGGAGCCT1160                                  GCGGCTTAATTTGACTCAACACGGGGAAACTCACCAGGTC1200                                  CAGACATGGGAAGGATTGACAGATTGATAGCTCTTTCTTG 1240                                 ATTCTATGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGG1280                                  AGTGATCTGTCTGGTTAATTTCGATAACGAACGAGACCTT1320                                  GGCCTGCTAAATAGGGTCGGTAACTTCGGTTATCGTATCA1360                                  CTTCTTAGAGGGACTTTGCGTGTCTAACGC AAGGAAGTTT1400                                 GAGGCAATAACAGGTCTGTGATGCCCTTAGATGTTCTGGG1440                                  CTGCACGCGCGCTACACTGATGCATGCAACGAGTTTTTAC1480                                  CTTGACCGACGGGGCTGGGTAATCTTCTGAGGGTGCATCG1520                                  TGATGGGGATAGATTATTGC AATTATTAGTCTTCAACGAG1560                                 GAATGCCTAGTAGGCGCAAGTCAGCAGCTTGCGCCGATTA1600                                  CGTCCCTGCCTCTTGTACACACCGCCCGTCGCTGCAACCG1640                                  ATCGGAGGGTCCTGTGAATTCATCGGACTGGCCATTCTCA1680                                  CTTTGGGGC TGGCCGGGAAGTTGCGTAAATAGAGCCCTCT1720                                 AAAGGATGCAAAAGTCGTAACACGGTTT1748                                              (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1744 bases                                                        (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      TAGTCATATGCTTGTCTCAAAGATTAAGCCATGCATGTCT40                                    AAGTATAAACTTTTATACGGTGAAACTGCGAATGGCTCAT80                                    TAAAACAGTTATAGTTTATTTGATGGTCATTTTTACATGG120                                   ATAACCATGGTAATTCTATGGCTAATACATGCGCA TAGGC160                                  TTCCTTCTTTGAAGGGGCTGTGTTTATTAGATACAAAACC200                                   AACCCACCTTGTGGAGCCTTGGTGATTCATAGTAACCGAA240                                   CGGATCGCAGTTGGCTTTCGGGCCCGCGATGGATCATTCA280                                   AGTTTCTGACCTATCAGCTTTCGACGGT AGGGTATTGGCC320                                  TACCGTGGCAGTGACGGGTAACGGGGAATTAGGGTTCGAT360                                   TCCGGAGAGGGAGCCTGAGAAACGGCTACCACATCTAAGG400                                   AAGGCAGCAGGCGCGCAAATTACCCAATGAAAACAGTTTC440                                   GAGGTAGTGACGAGAAATAA CAATACAGGGCATTTAATGC480                                  TTTGTAATTGGAATGATGGGAATGTAAAACCCTTCCAGAG520                                   TAACAATTGGAGGGCAAGTCTGGTGCCAGCAGCCGCGGTA560                                   ATTCCAGCTCCAATAGTGTATATTAGAGTTGTTGCAGTTA600                                   AAAAGCTCGTAGT TGGATTTCTGTCGTGGTCAGCCTGCGC640                                  TGCCCGTATGGGTGTGCGCGTGGTTTGCCCGCGGCTTTCT680                                   TCCGGTAGCCTCCGGCTCTTAATTGCGTCGGTGGGTGTTC720                                   TGGAACTTTTACTTTGAGAAAAATAGAGTGTTTCAAGCAG760                                   GCTTGT CGCCCTGAATACTGCAGCATGGAATAATAAGATA800                                  GGACCTCGGTTCTATTTTGTTGGTTTCTAGGACCAAGGTA840                                   ATGATTAATAGGGACAGTTGGGGGCATTCGTATTTAACTG880                                   TCAGAGGTGAAATTCTTAGATTTGTTAAAGACGAACTACT920                                   GCGAAAGCATTTGCCAAGGATGTTTTCATTAATCAAGAAC960                                   GACAGTAGGGGGTTTGAAGACGATTAGATACCGTCGTAAT1000                                  CTCTACCATAAACTATGCCGACTAGAGATAGGGAAATGCC1040                                  TACCTTGGCTTCTCCTGCACCTCATGAGAAATCAAAG TCT1080                                 CTGGGTTCTGGGGGGAGTATGGTCGCAAGGCTGAAACTTA1120                                  AAGGAATTGACGGAGGGGCACCACCAGGCGTGGAGCCTGC1160                                  GGCTTAATTTGACTCAACACGGGGAAACTCACCAGGTCCA1200                                  GACATGGGAAGGATTGACAGATTGAT AGCTCTTTCTTGAT1240                                 TCTATGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAG1280                                  TGATCTGTCTGGTTAATTTCGATAACGAACGAGACCTTGG1320                                  CCTGCTAAATAGGGTCGGTGACCTCGGTCACGCATCGCTT1360                                  CTTAGAGGGACTTTG CGTGTCTAACGCAGGGAAGTTCGAG1400                                 GCAATAACAGGTCTGTGATGCCCTTAGATGTTCTGGGCTG1440                                  CACGCGCGCTACACTGATGCATGCAACGAGTTCTTACCTT1480                                  GGCCGACGGGGCTGGGTAATCTTGTGGGGGTGCATCGTGA1520                                  TGGG GATAGATTATTGCAATTATTAGTCTTCAACGAGGAA1560                                 TGCCTAGTAGGCGCAAGTCAGCAGCTTGCGCCGATTACGT1600                                  CCCTGCCTCTTGTACACACCGCCCGTCGCTGCAACCGATG1640                                  GAGGGTCCTGTAAATTCATCGGACTGGCCAACCCCATTTT 1680                                 GGGGCTGGCTGGAAAGTTGCGTAAATAGAGCCCTCTAAAG1720                                  GATGCAAAAGTCGTAACACGGTTT1744                                                  (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1750 bases                                                        (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      TAGTCATATGCTTGTCTCAAAGATTAAGCCATGCATGTCT40                                    AAGTATAAACTTTTATACGGTGAAACTGCGAATGGCTCAT80                                    TAAAACAGTTATAGTTTATTTGATGGTCTTTTTTACATGG120                                   ATAACCATGGTAATTCTATG GCTAATACATGCGCAAAAGC160                                  TACCTTCTTTGGAGGAGCTGTGTTTATTAGATACAAAACC200                                   AGCCCACAATTCTTGTGGAGTCTTGGTGATTCATAGTAAC240                                   CGAACGGATCGCAGTTGGCTTTCGGGCCCGCGATGGATCA280                                   TTCAAGTTTCTG ACCTATCAGCTTTCGACGGTAGGGTATT320                                  GGCCTACCGTGGCAGTGACGGGTAACGGGGAATTAGGGTT360                                   CGATTCCGGAGAGGGAGCCTGAGAAACGGCTACCACATCT400                                   AAGGAAGGCAGCAGGCGCGCAAATTACCCAATGAAAACAG440                                   TTTCG AGGTAGTGACGAGAAATAACAATACAGGGCATTTT480                                  ATGCTTTGTAATTGGAATGATGGGAATGTAAAACCCTTCC520                                   AGAGTAACAATTGGAGGGCAAGTCTGGTGCCAGCAGCCGC560                                   GGTAATTCCAGCTCCAATAGTGTATATTAGAGTTGTTGCA600                                   GTTAAAAAGCTCGTAGTTGGATTTCTGTCGTGGTCAGCTT640                                   GCGCTGCCCGTATGGGTGTGCGCGTGGTTTGCCCTCGGCA680                                   TTCTTCCGGTAGCTTGTGGCGCTTAATTGCGTCTGCAAGT720                                   GCCCTGGAACTTTTACTTTGAGAAAAATAGAGTGTTTC AA760                                  GCAGGCTTGTCGCCCTGAATACTGCAGCATGGAATAATAG800                                   GATAGGACCTCGGTTCTATTTTGTTGGTTTCTAGGACCAA840                                   GGTAATGATTAATAGGGACAGTTGGGGGCATTCGTATTTA880                                   ACTGTCAGAGGTGAAATTCTTAGATTTGTT AAAGACGAAC920                                  TACTGCGAAAGCATTTGCCAAGGATGTTTTCATTAATCAA960                                   GAACGACAGTAGGGGGTTTGAAGACGATTAGATACCGTCG1000                                  TAATCTCTACCATAAACTATGCCGACTAGAGATAGGAAAA1040                                  CGCCTCCCTTGGCTTCTCCTG GACCTCATGAGAAATCAAA1080                                 GTCTCTGGGTTCTGGGGGGAGTATGGTCGCAAGGCTGAAA1120                                  CTTAAAGGAATTGACGGAGGGGCACCACCAGGCGTGGAGC1160                                  CTGCGGCTTAATTTGACTCAACACGGGGAAACTCACCAGG1200                                  TCCAGACATG GGAAGGATTGACAGATTGATAGCTCTTTCC1240                                 TGATTCTATGGGTGGTGGTGCATGGCCGTTCTTAGTTGGT1280                                  GGAGTGATTTGTCTGGTTAATTTCGATAACGAACGAGACC1320                                  TTGGCCTGCTAAATAGGGTCGGTGACCTCGGTTACCGTAT1360                                   CACTTCTTAGAGGGACATTGCGTGTCTAACGCAAGGAAGT1400                                 TTGAGGCAATAACAGGTCTGTGATGCCCTTAGATGTTCTG1440                                  GGCTGCACGCGCGCTACACTGATGCATGCAACGAGTTTTC1480                                  ACCTTGTCCGATGGGGCTGGGTAATCTTGTGAGGGTG CAT1520                                 CGTGATGGGGATAGATTATTGCAATTATTAGTCTTCAACG1560                                  AGGAATGCCTAGTAGGCGCAAGTCAGCAGCTTGTGCCGAT1600                                  TACGTCCCTGCCTCTTGTACACACCGCCCGTCGCTGCAAC1640                                  CGATCGGAGGGTCCTGTAAATTCATC GGACTGACCAGCCC1680                                 CAACTTGGGGCTGGTCGGAAAGTTGCGTAAATAGAGCCCT1720                                  CTAAAGGATGCAAAAGTCGTAACACGGTTT1750                                            (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1749 bases                                                        (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      TAGTCATATGCTTGTCTCAAAGATTAAGCCATGCATGTCT40                                    AAGTATAAGCTTTTATACGGTGAAACTGCGAATGGCTCAT80                                    TAAAACAGTTATAGTTTATTTGATGGTCTTTTTTACATGG120                                   ATAA CCATGGTAATTCTATGGCTAATACATGCGCATAGGC160                                  CTCCTCCTCTGGAGGGGCTGTGTTTATTAGCTACAAAACC200                                   AACCCACTTTTGTGGAGCCTTGGTGATTCATAGTAACCGA240                                   ACGGATCGCAGTTGGCTTTCGGGCCCGCGATGGATCATTC28 0                                  AAGTTTCTGACCTATCAGCTTTCGACGGTAGGGTATTGGC320                                   CTACCGTGGCAGTGACGGGTAACGGGGAATTAGGGTTCGA360                                   TTCCGGAGAGGGAGCCTGAGAAACGGCTACCACATCTAAG400                                   GAAGGCAGCAGGCGCGCAAATTACCCAATGAAAACAG TTT440                                  CGAGGTAGTGACGAGAAATAACAATACAGGGCATTTTATG480                                   CTTTGTAATTGGAATGATGGGAATGTAAAACCCTTCCAGA520                                   GTAACAATTGGAGGGCAAGTCTGGTGCCAGCAGCCGCGGT560                                   AATTCCAGCTCCAATAGTGTATATTAGAGT TGTTGCAGTT600                                  AAAAAGCTCGTAGTTGGATTTCTGTCGTGGTCTTCCTGTG640                                   CTGCCCGTATCGGTGCACGTGGCTTGCCCTCGACTTTCTT680                                   CCGGTAGCCTCCTGCGCTTCACTGCGTGGGCTGGTGTTCT720                                   GGAACTTTTACTTTGAGAAAAA TAGAGTGTTTCAAGCAGC760                                  TTGTCGCCCTGAATACTGCAGCATGGAATAATAAGATAGG800                                   ACCTCGGTTCTATTTTGTTGGTTTCTAGGACCAAGGTAAT840                                   GATTAATAGGGACAGTTGGGGGCATTCGTATTTAACTGTC880                                   AGAGGTGAAATTCTT AGATTTGTTAAAGACGAACTACTGC920                                  GAAAGTTTGCCAAGGATGTTTTCATTAATCAAGAACGACA960                                   GTAGGGGGTTTGAAGACGATTAGATACCGTCGTAATCTCT1000                                  ACCATAAACTATGCCGACTAGAGATAGGGAAACGCCTACC1040                                  TTGGCT TCTCCTGCACCTCATGAGAAATCAAAGTCTCTGG1080                                 GTTCTGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAGG1120                                  AATTGACGGAGGGGCACCACCAGGCGTGGAGCCGGGCCTT1160                                  AATTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAT1 200                                 GGGAAGGATTGACAGATTGATAGCTCTTTCTTGATTCTAT1240                                  GGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATC1280                                  TGTCTGGTTAATTTCGATAACGAACGAGACCTTGGCCTGC1320                                  TAAATAGGGTCGGTGACCCTGGGTCACCAGGT CACCGCAT1360                                 CGCTTCTTAGAGGAACTTTGCGTGTCTAACGCAAGGAAGT1400                                  TTGAGGCAATAACAGGTCTGTGATGCCCTTAGATGTTCTG1440                                  GGCTGCACGCGCGCTACACTGATGCATGCAACGAGTTTTT1480                                  ACCTTGCCCGATGGGCGTGGG TAATCTTGTGAGGGTGCAT1520                                 CGTGATGGGGATAGATTATTGCAATTATTAGTCTTCAACG1560                                  AGGAATGCCTAGTAGGCGCAAGTCAGCAGCTTGCGCCGAC1600                                  TAAGTCCCTGCCTCTTGTACACACCGCCCGTCGCTGCAAC1640                                  CGATCGGAGG GTCCTGTGAATTCATCGGATGGCCATCCCC1680                                 TTCTTGGGGCTGGCCGGGAAGTTGCGTAAATAGAGCCCTC1720                                  TAAAGGATGCAAAAGTCGTAACACGGTTT1749                                             (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1756 bases                                                         (B) TYPE: nucleic acid                                                       (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      TAGTCATATGCTTGTCTCAAAGATTAAGCCATGCATGTCT40                                    AAGTATAAGCTTTTATACGGTGAAACTGCGAATGGCTCAT80                                    TAAAACAGTTATAGTTTATTTGATGGTCTCATTTTA CATG120                                  GATAACCATGGTAATTCTATGGCTAATACATGCGCAAAGG160                                   TCACCTCCTTTGGAGGGGCTGTGTTTATTAGATACAAAAC200                                   CAACCCACTTAACGGTGGAGCCTTGGTGATTCATAGTAAC240                                   CGAACGGATCGCAGTTGGTTCTTTTGGAC CCGCGATGGAT280                                  CATTCAAGTTTCTGACCTATCAGCTTTCGACGGTAGGGTA320                                   TTGGCCTACCGTGGCAGTGACGGGTAACGGGGAATTAGGG360                                   TTCGATTCCGGAGAGGGAGCCTGAGAAACGGCTACCACAT400                                   CTAAGGAAGGCAGCAGGCGCG CAAATTACCCAATGAAAAC440                                  AGCTTCGAGGTAGTGACGAGAAATAACAATACAGGGCATT480                                   TTATGCTTTGTAATTGGAATGATGGAAATGTAAAACCCTT520                                   CCAGAGTAACAATTGGAGGGCAAGTCTGGTGCCAGCAGCC560                                   GCGGTAATTCCGGC TCCAATAGTGTATATTAGAGTTGTTG600                                  CAGTTAAAAAGCTCGTAGTTGGATTTCTGTCGTGGTCATC640                                   CGGCGCCGCCCGTATGGGTGTGGGCCTGGCATGCCCTCGG680                                   CTTATTTCCGGTAGCCTTCCGCGCTTAATTGCGTGTGTTG720                                   GTGTTCT GGAACTTTTACTTTGAGAAAAATAGAGTGTTTC760                                  AAGCAGGCTTGTCGCCCTGAATACTGCAGCATGGAATAAT800                                   AAGATAGGACCTCGGTTCTATTTTGTTGGTTTCTAGGACC840                                   AAGGTAATGATTAATAGGGACAGTTGGGGGCATTTGTATT880                                    TAACTGTCAGAGGTGAAATTCTTAGATTTGTTAAAGACGA920                                  ACTACTGCGAAAGCATTTGCCAAGGATGTTTTCATTAATC960                                   AAGAACGACAGTAGGGGGTTTGAAGACGATTAGATACCGT1000                                  CGTAATCTCTACCATAAACTATGCCGACTAGAGATAGGG A1040                                 AACGCCTACCTTGGCTTCTCCTGCACCTCATGAGAAATCA1080                                  AAGTCTCTGGGTTCTGGGGGGAGTATGGTCGCAAGGCTGA1120                                  AACTTAAAGGAATTGACGGAGGGGCACCACCAGGCGTGGA1160                                  GCCTGCGGCTTAATTTGACTCAACACGG GGAAACTCACCA1200                                 GGTCCAGACATGGGAAGGATTGACAGATTGATAGCTCTTT1240                                  CTTGATTCTATGGGTGGTGGTGCATGGCCGTTCTTAGTTG1280                                  GTGGAGTGATCTGTCTGGTTAATTTCGATAACGAACGAGA1320                                  CCTTAGCCTGCTAAATA GGGTCAGTAACTTCACGATTACT1360                                 GTATCACTTCTTAGAGGGACTTTGCGTGTCTAACGCAAGG1400                                  AAGTTTGAGGCAATAACAGGTCTGTGATGCCCTTAGATGT1440                                  TCTGGGCTGCACGCGCGCTACACTGATGCATGCAACGAGT1480                                  TTTTAC CTTGGCCGGCAGGTCTGGGTAATCTTTTGAGTGT1520                                 GCGTCGTGATGGGGATAAATTATTGCAATTATTAATCTTC1560                                  AACGAGGAATGCCTAGTAGGCGCAAGTCAGCAGCTTGCGC1600                                  CGATTAAGTCCCTGCCTCTTGTACACACCGCCCGTCGCTG1 640                                 CAACCGATCGGAGGGTCCTGTGAATTCATCGGACGGACAA1680                                  GCCTTACTTTGTGGGGCTGGTCGGGAAGTTGCGTAAATAG1720                                  AGCCCTCTAAAGGATGCAAAAGTCGTAACACGGTTT1756                                      (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 ( A) LENGTH: 1747 bases                                                       (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      TAGTCATATGCTTGTCTCAAAGATTAAGCCATGCATGTCT40                                    AAGTATAAGCTTTTATACGGTGAAACTGCGAATGGCTCAT80                                    TAAAACAGTTATAGTTTATT TGATGGTCTTTTTTACATGG120                                  ATAACCATGGTAATTCTATGGCTAATACATACGCAAAGGC160                                   TACCTTCTCTGGAGGGGCTGTGTTTATTAGATACAAAACC200                                   AACCCACTTTTGTGGAGTCATGGTGATTCATAGTAACCGA240                                   ACGGATCGCAGTT GGCTTTCGGGCCCGCGATGGATCATTC280                                  AAGTTTCTGACCTATCAGCTTTCGACGGTAGGGTATTGGC320                                   CTACCGTGGCAGTGACGGGTAACGGGGAATTAGGGTTCGA360                                   TTCCGGAGAGGGAGCCTGAGAAACGGCTACCACATCTAAG400                                   GAAGGC AGCAGGCGCGCAAATTACCCAATGAAAACAGTTT440                                  CGAGGTAGTGACGAGAAATAACAATACAGGGCATTTTATG480                                   CTTTGTAATTGGAATGATGGGAATGTAAAACCCTTCCAGA520                                   GTAACAATTGGAGGGCAAGTCTGGTGCCAGCAGCCGCGGT560                                   AATTCCAGCTCCAATAGTGTATATTAGAGTTGTTGCAGTT600                                   AAAAAGCTCGTAGTTGGATTTCTGTCGTGGTCAGCCTGCG640                                   CTGCCCGTATGGGTGTGCGCGTGGTTTGCCCTCGGCATAT680                                   TTCTGGTAGCCTCTGGCGCTTTATTGCGTTGGTAGGTGT T720                                  CTGGAACTTTTACTTTGAGAAAAATAGAGTGTTTCAAGCA760                                   GGCTTGTCGCCCTGAATACTGCAGCATGGAATAATAAGAT800                                   AGGACCTCGGTTCTATTTTGTTGGTTTCTAGGACCAAGGT840                                   AATGATTAATAGGGACAGTTGGGGGCATTCG TATTTAACT880                                  GTCAGAGGTGAAATTCTTAGATTTGTTAAAGACGAACTAC920                                   TGCGAAAGCATTTGCCAAGGATGTTTTCATTAATCAAGAA960                                   CGACAGTAGGGGGTTTGAAGACGATTAGATACCGTCGTAA1000                                  TCTCTACCATAAACTATGCCGAC TAGAGATAGGGAAATGC1040                                 CTACCTTGGCTTCTCCTGCACCTCATGAGAAATCAAAGTC1080                                  TCTGGGTTCTGGGGGGAGTATGGTCGCAAGGCTGAAACTT1120                                  AAAGGAATTGACGGAGGGGCACCACCAGGCGTGGAGCCTG1160                                  CGGCTTAATTTG ACTCAACACGGGGAAACTCACCAGGTCC1200                                 AGACATGGGAAGGATTGACAGATTGATAGCTCTTTCTTGA1240                                  TTCTATGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGA1280                                  GTGATCTGTCTGGTTAATTTCGATAACGAACGAGACCTTG1320                                  G CCTGCTAAATAGGGTCAGTAACTTCGGTTACTGTATCAC1360                                 TTCTTAGAGGGACTTTACGTGTCTAACGCAAGGAAGTTTG1400                                  AGGCAATAACAGGTCTGTGATGCCCTTAGATGTTCTGGGC1440                                  CGCACGCGCGCTACACTGATGCATGCAACGAGTTTTTAC C1480                                 TTGCCCGATGGGGCTGGGTAATCTTGTGAGGGTGCATCGT1520                                  GATGGGGATAGATTATTGCAATTATTAGTCTTCAACGAGG1560                                  AATGCCTAGTAGGCGCAAGTCAGCAGCTTGCGCCGACTAC1600                                  GTCCCTGCCCCTTGTACACACCGCCCGT CGCTGCAACCGA1640                                 TCGGAGGGTCCTGTGAATTCATCGGACTGGCCAACCCCAC1680                                  TTTGGGGCTGGCCGGGAAGTTGCGTAAATAGAGCCCTCTA1720                                  AAGGATGCAAAAGTCGTAACACGGTTT1747                                               (2) INFORMATION FOR SEQ ID NO:30:                                             ( i) SEQUENCE CHARACTERISTICS:                                                (A) LENGTH: 1756 bases                                                        (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                      TAGTCATATGCTTGTCTCAAAGATTAAGCCATGCATGTCT40                                    AAGTATAAGCTTTTATACGGTGAAACTGCGAATGGCTCAT80                                    TAAAA CAGTTATAGTTTATTTGATGGTCTCATTTTACATG120                                  GATAACCATGGTAATTCTATGGCTAATACATGCGCAAAGG160                                   TCACCTCCTTTGGAGGGGCTGTGTTTATTAGATACAAAAC200                                   CAACCCACTTTGTAGTGGAGTCTTGGTGATTCATAGTAAC240                                   CGAACGGATCGCAGTTGGTTCTTTTGGGCCCGCGATGGAT280                                   CATTCAAGTTTCTGACCTATCAGCTTTCGACGGTAGGGTA320                                   TTGGCCTACCGTGGCAGTGACGGGTAACGGGGAATTAGGG360                                   TTCGATTCCGGAGAGGGAGCCTGAGAAACGGCTACCAC AT400                                  CTAAGGAAGGCAGCAGGCGCGCAAATTACCCAATGAAAAC440                                   AGCTTCGAGGTAGTGACGAGAAATAACAATACAGGGCATT480                                   TTATGCTTTGTAATTGGAATGATGGGAATGTAAAACCCTT520                                   CCAGAGTAACAATTGGAGGGCAAGTCTGGT GCCAGCAGCC560                                  GCGGTAATTCCAGCTCCAATAGTGTATATTAGAGTTGTTG600                                   CAGTTAAAAAGCTCGTAGTTGGATTTCTGTCGTGGTCATC640                                   CGGCGTCGCCCGTATGGGTGTGTGCCTGGCATGCCCTCGG680                                   CTTATTTCCGGTAGCCTTCCGCG CTTAATTGCGTGTGTTG720                                  GTGTTCTGGAACTTTTACTTTGAGAAAAATAGAGTGTTTC760                                   AAGCAGGCTTGTCGCCCTGAATACTGCAGCATGGAATAAT800                                   AAGATAGGACCTCGGTTCTATTTTGTTGGTTTCTAGGACC840                                   AAGGTAATGATTAATA GGGACAGTTGGGGGCATTCGTATT880                                  TAACTGTCAGAGGTGAAATTCTTAGATTTGTTAAAGACGA920                                   ACTACTGCGAAAGCATTTGCCAAGGATGTTTTCATTAATC960                                   AAGAACGACAGTAGGGGGTTTGAAGACGATTAGATACCGT1000                                  CGTAATCT CTACCATAAACTATGCCGACTAGAGATAGGGA1040                                 AACGCCTACCTTGGCTTCTCCTGCACCTCATGAGAAATCA1080                                  AAGTCTCTGGGTTCTGGGGGGAGTATGGTCGCAAGGCTGA1120                                  AACTTAAAGGAATTGACGGAGGGGCACCACCAGGCGTGGA116 0                                 GCCTGCGGCTTAATTTGACTCAACACGGGGAAACTCACCA1200                                  GGTCCAGACATGGGAAGGATTGACAGATTGATAGCTCTTT1240                                  CTTGATTCTATGGGTGGTGGTGCATGGCCGTTCTTAGTTG1280                                  GTGGAGTGATCTGTCTGGTTAATTTCGATAACGA ACGAGA1320                                 CCTTAGCCTGCTAAATAGGGTCAGTAACTTTGCGGTTACT1360                                  GTATCACTTCTTAGAGGGACTTTGCGTGTCTAACGCAAGG1400                                  AAGTTTGAGGCAATAACAGGTCTGTGATGCCCTTAGATGT1440                                  TCTGGGCTGCACGCGCGCTACAC TGATGCATGCAACGAGT1480                                 TTTTACCTTGGCCGACAGGTCTGGGTAATCTTTTGAGTGT1520                                  GCATCGTGATGGGGATAGATTATTGCAATTATTAATCTTC1560                                  AACGAGGAATGCCTAGTAGGCGCAAGTCAGCAGCTTGCGC1600                                  CGATTAAGTCCC TGCCTCTTGTACACACCGCCCGTCGCTG1640                                 CAACCGATCGGAGGGTCCTGTGAATTCATCGGACGGACAA1680                                  GCCTTACTTTGTGGGGCTGGTCGGGAAGTTGCGTAAATAG1720                                  AGCCCTCTAAAGGATGCAAAAGTCGTAACACAGTTT1756                                       (2) INFORMATION FOR SEQ ID NO:31:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                      GTCGCAAGGCTGAAAC16                                                            (2) INFORMATION FOR SEQ ID NO:32:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 bases                                                          (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                      CTTGCGCCTACTAGGC16                                                            (2) INFORMATION FOR SEQ ID NO:33:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                      GGGGGGAGTATGGTCTGCA AGGC23                                                    (2) INFORMATION FOR SEQ ID NO:34:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                      GCATGCATCAGTGTAGCTGCGCG23                                                     (2) INFORMATION FOR SEQ ID NO:35:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 bases                                                           (B) TYPE: nucleic acid                                                       (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                      TACGATAACCGAAGTTACCG20                                                        (2) INFORMATION FOR SEQ ID NO:36:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                      GATACGGTAACCAAAGTCACC21                                                       (2) INFORMATION FOR SEQ ID NO:37:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                      GATACGGTAACCGAGGTCA19                                                         (2) INFORMATION FOR SEQ ID NO:38:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 20 bases                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                      CAAAACCAACCCACTTAACG20                                                        (2) INFORMATION FOR SEQ ID NO:39:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      ( D) TOPOLOGY: linear                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                      TGATACAGTAACCGAAGTTACTG23                                                     (2) INFORMATION FOR SEQ ID NO:40:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                      TACAGTAACCGCAAAGTTACTG22                                                      (2 ) INFORMATION FOR SEQ ID NO:41:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                      GGTCTCGTTCGTTAATCGAA20                                                        (2) INFORMATION FOR SEQ ID NO:42:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 bases                                                          (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                      CATCACAGACCTGTTATTGCC21                                                       (2) INFORMATION FOR SEQ ID NO:43:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                      CATAGAACGGC CATGCA17                                                          (2) INFORMATION FOR SEQ ID NO:44:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                      AAACTTAAAGGAATTGACGG20                                                        (2) INFORMATION FOR SEQ ID NO:45:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 bases                                                           (B) TYPE: nucleic acid                                                       (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                      CGGTGTGTACAAAGGGCAGG20                                                        (2) INFORMATION FOR SEQ ID NO:46:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                      AACTTCGGTTATCGTATCACTTC23                                                     (2) INFORMATION FOR SEQ ID NO:47:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                                      GTAACTTCGGTTATCGTATCACT23                                                     (2) INFORMATION FOR SEQ ID NO:48:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 21 bases                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                                      CTTCGGTTATCGTATCACTTC21                                                       (2) INFORMATION FOR SEQ ID NO:49:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                                      ACTTCGGTTATCGTATCACTT21                                                       (2) INFORMATION FOR SEQ ID NO:50:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 508 bases                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                                      AAACTTAAAGGAATTGACGGAGGGGCAC CACCAGGCGTGG40                                   AGCCTGCGGCTTAATTTGACTCAACACGGGGAAACTCACC80                                    AGGTCCAGACATGGGAAGGATTGACAGATTGATAGCTCTT120                                   TCTTGATTCTATGGGTGGTGGTGCATGGCCGTTCTTAGTT160                                   GGTGGAGTGATCTGTCT GGTTAATTTCGATAACGAACGAG200                                  ACCTTGGCCTGCTAAATAGGGTCGGTGACTTTGGTTACCG240                                   TATCGCTTCTTAGAGGGACTTTGCGTGTCTAACGCAAGGA280                                   AGTTTGAGGCAATAACAGGTCTGTGATGCCCTTAGATGTT320                                   CTGGGC TGCACGCGCGCTACACTGATGCATGCAACGAGTT360                                  TTTACCTTGACCGACGGGGCTGGGTAATCTTGTGAGGGTG400                                   CATCGTGATGGGGATAGATTATTGCAATTATTAGTCTTCA440                                   ACGAGGAATGCCTAGTAGGCGCAAGTCAGCACTTGCGCCG 480                                  ATTACGTCCCTGCCCTTTGTACACACCG508                                           

What is claimed is:
 1. A DNA probe, the DNA probe consisting of 5'CAAGACTCCACAAGAATTGTG 3' (SEQ ID NO:11).
 2. A composition, consistingessentially of a DNA probe consisting of 5' CAAGACTCCACAAGAATTGTG 3'(SEQ ID NO: 11).